Diagnosis and treatment of malignant neoplasms

ABSTRACT

The invention features a method for diagnosing a malignant neoplasm in a mammal by contacting a bodily fluid from the mammal with an antibody which binds to an human aspartyl (asparaginyl) beta-hydroxylase (HAAH) polypeptide and methods of treating malignant neoplasms by inhibiting HAAH.

This application is a continuation of U.S. patent application Ser. No.09/903,023, filed Jul. 11, 2001, which is a division of U.S. patentapplication Ser. No. 09/436,184, filed Nov. 8, 1999, each of which isherein incorporated by reference in its entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with U.S. Government support under NationalInstitutes of Health grants CA-35711, AA-02666, AA-02169, and AA11431.The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Primary malignant central nervous system (CNS) neoplasms, particularlyglioblastomas, are highly fatal due to their aggressive and widespreadinfiltration of the brain and resistance to anti-cancer treatments.Although progress has been made in unraveling the pathologicalmechanisms underlying CNS cancers as well as other cancer types, tumorspecific therapeutic approaches and methods of diagnosis have beenlargely elusive.

SUMMARY OF THE INVENTION

The invention features a method for diagnosing a malignant neoplasm in amammal by contacting a bodily fluid from the mammal with an antibodywhich binds to an human aspartyl (asparaginyl) beta-hydroxylase (HAAH)polypeptide under conditions sufficient to form an antigen-antibodycomplex and detecting the antigen-antibody complex. Malignant neoplasmsdetected in this manner include those derived from endodermal tissue,e.g., colon cancer, breast cancer, pancreatic cancer, liver cancer, andcancer of the bile ducts. Neoplasms of the central nervous system (CNS)such as primary malignant CNS neoplasms of both neuronal and glial cellorigin and metastatic CNS neoplasms are also detected. Patient derivedtissue samples, e.g., biopsies of solid tumors, as well as bodily fluidssuch as a CNS-derived bodily fluid, blood, serum, urine, saliva, sputum,lung effusion, and ascites fluid, are contacted with an HAAH-specificantibody.

The assay format is also useful to generate temporal data used forprognosis of malignant disease. A method for prognosis of a malignantneoplasm of a mammal is carried out by (a) contacting a bodily fluidfrom the mammal with an antibody which binds to an HAAH polypeptideunder conditions sufficient to form an antigen-antibody complex anddetecting the antigen-antibody complex; (b) quantitating the amount ofcomplex to determine the level of HAAH in the fluid; and (c) comparingthe level of HAAH in the fluid with a normal control level of HAAH. Anincreasing level of HAAH over time indicates a progressive worsening ofthe disease, and therefore, an adverse prognosis.

The invention also includes an antibody which binds to HAAH. Theantibody preferably binds to a site in the carboxyterminal catalyticdomain of HAAH. Alternatively, the antibody binds to an epitope that isexposed on the surface of the cell. The antibody is a polyclonalantisera or monoclonal antibody. The invention encompasses not only anintact monoclonal antibody, but also an immunologically-active antibodyfragment, e. g. , a Fab or (Fab)₂ fragment; an engineered single chainFv molecule; or a chimeric molecule, e.g., an antibody which containsthe binding specificity of one antibody, e.g., of murine origin, and theremaining portions of another antibody, e.g., of human origin.Preferably the antibody is a monoclonal antibody such as FB50, 5C7, 5E9,19B, 48A, 74A, 78A, 86A, HA238A, HA221, HA 239, HA241, HA329, or HA355.Antibodies which bind to the same epitopes as those monoclonalantibodies are also within the invention.

An HAAH-specific intrabody is a recombinant single chain HAAH-specificantibody that is expressed inside a target cell, e.g., tumor cell. Suchan intrabody binds to endogenous intracellular HAAH and inhibits HAAHenzymatic activity or prevents HAAH from binding to an intracellularligand. HAAH-specific intrabodies inhibit intracellular signaltransduction, and as a result, inhibit growth of tumors whichoverexpress HAAH.

A kit for diagnosis of a tumor in a mammal contains an HAAH-specificantibody. The diagnostic assay kit is preferentially formulated in astandard two-antibody binding format in which one HAAH-specific antibodycaptures HAAH in a patient sample and another HAAH-specific antibody isused to detect captured HAAH. For example, the capture antibody isimmobilized on a solid phase, e.g., an assay plate, an assay well, anitrocellulose membrane, a bead, a dipstick, or a component of anelution column. The second antibody, i.e., the detection antibody, istypically tagged with a detectable label such as a calorimetric agent orradioisotope.

Also within the invention is a method of inhibiting tumor growth in amammal, which is carried out by administering to the mammal a compoundwhich inhibits expression or enzymatic activity of HAAH. Preferably, thecompound is substantially pure nucleic acid molecule such as an HAAHantisense DNA, the sequence of which is complementary to a codingsequence of HAAH. Expression of HAAH is inhibited by contactingmammalian cells, e.g., tumor cells, with HAAH antisense DNA or RNA,e.g., a synthetic HAAH antisense oligonucleotide. For example, HAAHantisense nucleic acid is introduced into glioblastoma cells or othertumor cells which overexpress HAAH. Binding of the antisense nucleicacid to an HAAH transcript in the target cell results in a reduction inHAAH production by the cell. By the term “antisense nucleic acid” ismeant a nucleic acid (RNA or DNA) which is complementary to a portion ofan mRNA, and which hybridizes to and prevents translation of the mRNA.Preferably, the antisense DNA is complementary to the 5′ regulatorysequence or the 5′ portion of the coding sequence of HAAH mRNA (e.g., asequence encoding a signal peptide or a sequence within exon 1 of theHAAH gene). Standard techniques of introducing antisense DNA into thecell may be used, including those in which antisense DNA is a templatefrom which an antisense RNA is transcribed. The method is to treattumors in which expression of HAAH is upregulated, e.g., as a result ofmalignant transformation of the cells. The length of the oligonucleotideis at least 10 nucleotides and may be as long as the naturally-occurringHAAH transcript. Preferably, the length is between 10 and 50nucleotides, inclusive. More preferably, the length is between 10 and 20nucleotides, inclusive.

By “substantially pure DNA or RNA” is meant that the nucleic acid isfree of the genes which, in the naturally-occurring genome of theorganism from which the DNA of the invention is derived, flank a HAAHgene. The term therefore includes, for example, a recombinant nucleicacid which is incorporated into a vector, into an autonomouslyreplicating plasmid or virus, or into the genomic DNA of a procaryote oreucaryote at a site other than its natural site; or which exists as aseparate molecule (e.g., a cDNA or a genomic or cDNA fragment producedby PCR or restriction endonuclease digestion) independent of othersequences. It also includes a recombinant nucleic acid which is part ofa hybrid gene encoding additional polypeptide sequence such as a nucleicacid encoding an chimeric polypeptide, e.g., one encoding an antibodyfragment linked to a cytotoxic polypeptide. Alternatively, HAAHexpression is inhibited by administering a ribozyme or a compound whichinhibits binding of Fos or Jun to an HAAH promoter sequence.

Compounds, which inhibit an enzymatic activity of HAAH, are useful toinhibit tumor growth in a mammal. By enzymatic activity of HAAH is meanthydroxylation of an epidermal growth factor (EGF)-like domain of apolypeptide. For example an EGF-like domain has the consensus sequenceCX₇CX₄CX₁₀CXCX₈C (SEQ ID NO:1). HAAH hydroxylase activity is inhibitedintracellularly. For example, a dominant negative mutant of HAAH (or anucleic acid encoding such a mutant) is administered. The dominantnegative HAAH mutant contains a mutation which changes a ferrous ironbinding site from histidine of a naturally-occurring HAAH sequence to anon-iron-binding amino acid, thereby abolishing the hydroxylase activityof HAAH. The histidine to be mutated, e.g., deleted or substituted, islocated in the carboxyterminal catalytic domain of HAAH. For example,the mutation is located between amino acids 650-700 (such as the Hismotif, underlined sequence of SEQ ID NO:2) the native HAAH sequence. Forexample, the mutation is at residues 671, 675, 679, or 690 of SEQ IDNO:2. An HAAH-specific intrabody is also useful to bind to HAAH andinhibit intracellular HAAH enzymatic activity, e.g., by binding to anepitope in the catalytic domain of HAAH. Other compounds such asL-mimosine or hydroxypyridone are administered directly into a tumorsite or systemically to inhibit HAAH hydroxylase activity. TABLE 1 Aminoacid sequence of HAAH MAQRKNAKSS GNSSSSGSGS GSTSAGSSSP GARRETKHGG 61HKNGRKGGLS GTSFFTWFMV IALLGVWTSV AVVWFDLVDY EEVLGKLGIY DADGDGDFDV 121DDAKVLLGLK ERSTSEPAVP PEEAEPHTEP EEQVPVEAEP QNIEDEAKEQ IQSLLHEMVH 181AEHVEGEDLQ QEDGPTGEPQ QEDDEFLMAT DVDDRFETLE PEVSHEETEH SYHVEETVSQ 241DCNQDMEEMM SEQENPDSSE PVVEDERLHH DTDDVTYQVY EEQAVYEPLE NEGIEITEVT 301APPEDNPVED SQVIVEEVSI FPVEEQQEVP PETNRKTDDP EQKAKVKKKK PKLLNKFDKT 361IKAELDAAEK LRKRGKIEEA VNAFKELVRK YPQSPRARYG KAQCEDDLAE KRRSNEVLRG 421AIETYQEVAS LPDVPADLLK LSLKRRSDRQ QFLGHMRGSL LTLQRLVQLF PNDTSLKNDL 481GVGYLLIGDN DNAKKVYEEV LSVTPNDGFA KVHYGFILKA QNKIAESIPY LKEGIESGDP 541GTDDGRFYFH LGDAMQRVGN KEAYKWYELG HKRGHFASVW QRSLYNVNGL KAQPWWTPKE 601TGYTELVKSL ERNWKLIRDE GLAVMDKAKG LFLPEDENLR EKGDWSQFTL WQQGRRNENA 661CKGAPKTCTL LEKFPETTGC RRGQIKYSIM HPGTHVWPH T GPTNCRLRMH LGLVIPKEGC 721KIRCANETRT WEEGKVLIFD DSFEHEVWQD ASSFRLIFIV DVWHPELTPQ QRRSLPAI(SEQ ID NO: 2; GENBANK Accession No. S83325; His motif is underlined;conserved sequences within the catalytic domain are designated by boldtype)

For example, a compound which inhibits HAAH hydroxylation is apolypeptide that binds a HAAH ligand but does not transduce anintracellular signal or an polypeptide which contains a mutation in thecatalytic site of HAAH. Such a polypeptide contains an amino acidsequence that is at least 50% identical to a naturally-occurring HAAHamino acid sequence or a fragment thereof and which has the ability toinhibit HAAH hydroxylation of substrates containing an EGF-like repeatsequence. More preferably, the polypeptide contains an amino acidsequence that is at least 75%, more preferably at least 85%, morepreferably at least 95% identical to SEQ ID NO: .

A substantially pure HAAH polypeptide or HAAH-derived polypeptide suchas a mutated HAAH polypeptide is preferably obtained by expression of arecombinant nucleic acid encoding the polypeptide or by chemicallysynthesizing the protein. A polypeptide or protein is substantially purewhen it is separated from those contaminants which accompany it in itsnatural state (proteins and other naturally-occurring organicmolecules). Typically, the polypeptide is substantially pure when itconstitutes at least 60%, by weight, of the protein in the preparation.Preferably, the protein in the preparation is at least 75%, morepreferably at least 90%, and most preferably at least 99%, by weight,HAAH. Purity is measured by any appropriate method, e.g., columnchromatography, polyacrylamide gel electrophoresis, or HPLC analysis.Accordingly, substantially pure polypeptides include recombinantpolypeptides derived from a eucaryote but produced in E. coli or anotherprocaryote, or in a eucaryote other than that from which the polypeptidewas originally derived.

Nucleic acid molecules which encode such HAAH or HAAH-derivedpolypeptides are also within the invention. TABLE 2 HAAH cDNA sequencecggaccgtgc aatggcccag cgtaagaatg ccaagagcag 61 cggcaacagc agcagcagcggctccggcag cggtagcacg agtgcgggca gcagcagccc 121 cggggcccgg agagagacaaagcatggagg acacaagaat gggaggaaag gcggactctc 181 gggaacttca ttcttcacgtggtttatggt gattgcattg ctgggcgtct ggacatctgt 241 agctgtcgtt tggtttgatcttgttgacta tgaggaagtt ctaggaaaac taggaatcta 301 tgatgctgat ggtgatggagattttgatgt ggatgatgcc aaagttttat taggacttaa 361 agagagatct acttcagagccagcagtccc gccagaagag gctgagccac acactgagcc 421 cgaggagcag gttcctgtggaggcagaacc ccagaatatc gaagatgaag caaaagaaca 481 aattcagtcc cttctccatgaaatggtaca cgcagaacat gttgagggag aagacttgca 541 acaagaagat ggacccacaggagaaccaca acaagaggat gatgagtttc ttatggcgac 601 tgatgtagat gatagatttgagaccctgga acctgaagta tctcatgaag aaaccgagca 661 tagttaccac gtggaagagacagtttcaca agactgtaat caggatatgg aagagatgat 721 gtctgagcag gaaaatccagattccagtga accagtagta gaagatgaaa gattgcacca 781 tgatacagat gatgtaacataccaagtcta tgaggaacaa gcagtatatg aacctctaga 841 aaatgaaggg atagaaatcacagaagtaac tgctccccct gaggataatc ctgtagaaga 901 ttcacaggta attgtagaagaagtaagcat ttttcctgtg gaagaacagc aggaagtacc 961 accagaaaca aatagaaaaacagatgatcc agaacaaaaa gcaaaagtta agaaaaagaa 1021 gcctaaactt ttaaataaatttgataagac tattaaagct gaacttgatg ctgcagaaaa 1081 actccgtaaa aggggaaaaattgaggaagc agtgaatgca tttaaagaac tagtacgcaa 1141 ataccctcag agtccacgagcaagatatgg gaaggcgcag tgtgaggatg atttggctga 1201 gaagaggaga agtaatgaggtgctacgtgg agccatcgag acctaccaag aggtggccag 1261 cctacctgat gtccctgcagacctgctgaa gctgagtttg aagcgtcgct cagacaggca 1321 acaatttcta ggtcatatgagaggttccct gcttaccctg cagagattag ttcaactatt 1381 tcccaatgat acttccttaaaaaatgacct tggcgtggga tacctcttga taggagataa 1441 tgacaatgca aagaaagtttatgaagaggt gctgagtgtg acacctaatg atggctttgc 1501 taaagtccat tatggcttcatcctgaaggc acagaacaaa attgctgaga gcatcccata 1561 tttaaaggaa ggaatagaatccggagatcc tggcactgat gatgggagat tttatttcca 1621 cctgggggat gccatgcagagggttgggaa caaagaggca tataagtggt atgagcttgg 1681 gcacaagaga ggacactttgcatctgtctg gcaacgctca ctctacaatg tgaatggact 1741 gaaagcacag ccttggtggaccccaaaaga aacgggctac acagagttag taaagtcttt 1801 agaaagaaac tggaagttaatccgagatga aggccttgca gtgatggata aagccaaagg 1861 tctcttcctg cctgaggatgaaaacctgag ggaaaaaggg gactggagcc agttcacgct 1921 gtggcagcaa ggaagaagaaatgaaaatgc ctgcaaagga gctcctaaaa cctgtacctt 1981 actagaaaag ttccccgagacaacaggatg cagaagagga cagatcaaat attccatcat 2041 gcaccccggg actcacgtgtggccgcacac agggcccaca aactgcaggc tccgaatgca 2101 cctgggcttg gtgattcccaaggaaggctg caagattcga tgtgccaacg agaccaggac 2161 ctgggaggaa ggcaaggtgctcatctttga tgactccttt gagcacgagg tatggcagga 2221 tgcctcatct ttccggctgatattcatcgt ggatgtgtgg catccggaac tgacaccaca 2281 gcagagacgc agccttccagcaatttagca tgaattcatg caagcttggg aaactctgga gaga(SEQ ID NO:3; GENBANK Accession No. S83325; codon encoding initiatingmethionine is underlined).

Methods of inhibiting tumor growth also include administering a compoundwhich inhibits HAAH hydroxylation of a NOTCH polypeptide. For example,the compound inhibits hydroxylation of an EGF-like cysteine-rich repeatsequence in a NOTCH polypeptide, e.g., one containing the consensussequence CDXXXCXXKXGNGXCDXXCNNAACXXDGXDC (SEQ ID NO:4). Polypeptidescontaining an EGF-like cysteine-rich repeat sequence are administered toblock hydroxylation of endogenous NOTCH.

Growth of a tumor which overexpresses HAAH is also inhibited byadministering a compound which inhibits signal transduction through theinsulin receptor substrate (IRS) signal transduction pathway. Preferablythe compound inhibits IRS phosphorylation. For example, the compound isa peptide or non-peptide compound which binds to and inhibitsphosphorylation at residues 46, 465, 551, 612, 632, 662, 732, 941, 989,or 1012 of SEQ ID NO:5 . Compounds include polypeptides such those whichblock an IRS phosphorylation site such as a Glu/Tyr site. Antibodiessuch as those which bind to a carboxyterminal domain of IRS containing aphosphorylation site block IRS phosphorylation, and as a consequence,signal transduction along the pathway. Inhibition of IRS phosphorylationin turn leads to inhibition of cell proliferation. Other compounds whichinhibit IRS phosphorylation include vitamin D analogue EB1089 andWortmannin.

HAAH-overproducing tumor cells were shown to express HAAH bothintracellularly and on the surface of the tumor cell. Accordingly, amethod of killing a tumor cell is carried out by contacting such a tumorcell with a cytotoxic agent linked to an HAAH-specific antibody. TheHAAH-specific antibody (antibody fragment, or ligand which binds toextracellular HAAH) directs the chimeric polypeptide to the surface ofthe tumor cell allowing the cytotoxic agent to damage or kill the tumorcell to which the antibody is bound. The monoclonal antibody binds to anepitope of HAAH such as an epitope exposed on the surface of the cell orin the catalytic site of HAAH. The cytotoxic composition preferentiallykills tumor cells compared to non-tumor cell.

Screening methods to identify anti-tumor agents which inhibit the growthof tumors which overexpress HAAH are also within the invention. Ascreening method used to determine whether a candidate compound inhibitsHAAH enzymatic activity includes the following steps: (a) providing aHAAH polypeptide, e.g., a polypeptide which contains the carboxyterminalcatalytic site of HAAH; (b) providing a polypeptide comprising anEGF-like domain; (c) contacting the HAAH polypeptide or the EGF-likepolypeptide with the candidate compound; and (d) determininghydroxylation of the EGF-like polypeptide of step (b). A decrease inhydroxylation in the presence of the candidate compound compared to thatin the absence of said compound indicates that the compound inhibitsHAAH hydroxylation of EGF-like domains in proteins such as NOTCH.

Anti-tumor agents which inhibit HAAH activation of NOTCH are identifiedby (a) providing a cell expressing HAAH; (b) contacting the cell with acandidate compound; and (c) measuring translocation of activated NOTCHto the nucleus of said cell. Translocation is measured by using areagent such as an antibody which binds to a 110 kDa activation fragmentof NOTCH. A decrease in translocation in the presence of the candidatecompound compared to that in the absence of the compound indicates thatthe compound inhibits HAAH activation of NOTCH, thereby inhibitingNOTCH-mediated signal transduction and proliferation ofHAAH-overexpressing tumor cells.

Nucleotide and amino acid comparisons described herein were carried outusing the Lasergene software package (DNASTAR, Inc., Madison, Wis.). TheMegAlign module used was the Clustal V method (Higgins et al., 1989,CABIOS 5(2):151-153). The parameter used were gap penalty 10, gap lengthpenalty 10.

Hybridization is carried out using standard techniques, such as thosedescribed in Ausubel et al. (Current Protocols in Molecular Biology,John Wiley & Sons, 1989). “High stringency” refers to nucleic acidhybridization and wash conditions characterized by high temperature andlow salt concentration, e.g., wash conditions of 65° C. at a saltconcentration of 0.1×SSC. “Low” to “moderate” stringency refers to DNAhybridization and wash conditions characterized by low temperature andhigh salt concentration, e.g., wash conditions of less than 60° C. at asalt concentration of at least 1.0×SSC. For example, high stringencyconditions include hybridization at 42° C. in the presence of 50%formamide; a first wash at 65° C. in the presence of 2×SSC and 1% SDS;followed by a second wash at 65° C. in the presence of 0.1%×SSC. Lowerstringency conditions suitable for detecting DNA sequences having about50% sequence identity to an HAAH gene sequence are detected by, forexample, hybridization at about 42° C. in the absence of formamide; afirst wash at 42° C., 6×SSC, and 1% SDS; and a second wash at 50° C.,6×SSC, and 1% SDS.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph showing colony formation induced by transienttransfection of NIH-3T3 cells with various AAH cDNAs. Colony formationwas induced by transient transfection with 10 μg DNA. In contrast, themutant murine AAH construct without enzymatic activity has notransforming activity. The data is presented as mean number oftransformed foci ±SEM.

FIG. 2 is a bar graph showing the results of a densitometric analysis ofa Western blot assay of proteins produced by various murine AAH stablytransfected cell clones. In clones 7 and 18, there was a modest increasein HAAH gene expression, while the overexpression was to a lesser degreein clone 16.

FIGS. 3A-B are bar graphs showing colony formation in soft agarexhibited by HAAH stably transfected clones compared to HAAH enzymaticactivity. FIG. 3A shows a measurement of murine AAH enzymatic activityin clones 7, 16 and 18, and FIG. 3B shows colony formation exhibited byclones 7, 16 and 18. Data is presented as mean number of colonies 10days after plating ±SEM. All three clones with modest increases in HAAHenzymatic activity, that correlated with protein expression, exhibitedanchorage independent growth.

FIG. 4 is a bar graph showing tumor formation in nude mice injected withtransfected clones overexpressing murine AAH. Tumor growth was assessedafter 30 days. Mean tumor weight observed in mice injected with clones7, 16 and 18 as compared to mock DNA transfected clone. All animalsinjected with clones overexpressing HAAH developed tumors.

FIGS. 5A-D are bar graphs showing increased AAH expression in PNET2(FIGS. 5A, 5C) and SH-Sy5y (FIG. 5B) cells treated with retinoic acid(FIGS. 5A, 5B) or phorbol ester myristate (PMA; FIG. 5C) to induceneurite outgrowth as occurs during tumor cell invasion. The cells weretreated with 10 μM retinoic acid or 100 nM PMA for 0, 1, 2, 3, 4, or 7days. Cell lysates were analyzed by Western blot analysis using anHAAH-specific monoclonal antibody to detect the 85 kDa AAH protein. Thelevels of immunoreactivity were measured by volume densitometry(arbitrary units). The graphs indicate the mean ±S.D. of resultsobtained from three separate experiments. In FIG. 5D, PNET2 cells weretreated for 24 hours with sub-lethal concentrations of H₂O₂ to induceneurite retraction. Viability of greater than 90% of the cells wasdemonstrated by Trypan blue dye exclusion. Similar results were obtainedfor SH-Sy5y cells.

FIG. 6 is a bar graph showing the effects of AAH over-expression on thelevels of anti-apoptosis (Bcl-2), cell cycle-mitotic inhibitor (p16 andp21/Waf1), and proliferation (proliferating cell nuclear antigen; PCNA)molecules. PNET2 neuronal cells were stably transfected with thefull-length human cDNA encoding AAH (pHAAH) or empty vector (pcDNA). AAHgene expression was under control of a CMV promoter. Western blotanalysis was performed with cell lysates prepared from cultures thatwere 70 to 80 percent confluent. Protein loading was equivalent in eachlane. Replicate blots were probed with the different antibodies. Bargraphs depict the mean S.D.'s of protein expression levels measured inthree experiments. All differences are statistically significant byStudent T-test analysis (P<0.01-P<0.001).

FIG. 7 is a diagram of showing the components of the IRS-1 signaltransduction pathway.

FIG. 8 is a line graph showing growth curves generated in cellsexpressing the antisense HAAH compared to controls expressing GFP.

FIG. 9 is a diagram of the functional domains of the hIRS-1 protein andstructural organization of the point mutants. All mutant and “wild type”hIRS-1 proteins construct contain a FLAG (F) epitope (DYKDDDDK; SEQ IDNO:7) at the C-terminus. PH and PTB indicate pleckstrin homology andphosphotyrosine binding, regions, respectively.

DETAILED DESCRIPTION

HAAH is a protein belonging to the (α-ketoglutarate dependentdioxygenase family of prolyl and lysyl hydroxylases which play a keyrole in collagen biosynthesis. This molecule hydroxylates aspartic acidor asparagine residues in EGF-like domains of several proteins in thepresence of ferrous iron. These EGF-like domains contain conservedmotifs, that form repetitive sequences in proteins such as clottingfactors, extracellular matrix proteins, LDL receptor, NOTCH homologuesor NOTCH ligand homologues.

The alpha-ketoglutarate-dependent dioxygenase aspartyl (asparaginyl)beta-hydroxylase (AAH) specifically hydroxylates one aspartic orasparagine residue in EGF-like domains of various proteins. The 4.3-kbcDNA encoding the human AspH (hAspH) hybridizes with 2.6 kb and 4.3 kbtranscripts in transformed cells, and the deduced amino acid sequence ofthe larger transcript encodes an protein of about 85 kDa. Both in vitrotranscription and translation and Western blot analysis also demonstratea 56-kDa protein that may result from posttranslational cleavage of thecatalytic C terminus.

An physiological function of AAH is the post-translationalbeta-hydroxylation of aspartic acid in vitamin K-dependent coagulationproteins. However, the abundant expression of AAH in several malignantneoplasms, and low levels of AAH in many normal cells indicate a rolefor this enzyme in malignancy. The AAH gene is also highly expressed incytotrophoblasts, but not syncytiotrophoblasts of the placenta.Cytotrophoblasts are invasive cells that mediate placental implantation.The increased levels of AAH expression in human cholangiocarcinomas,hepatocellular carcinomas, colon cancers, and breast carcinomas wereprimarily associated with invasive or metastatic lesions. Moreover,overexpression of AAH does not strictly reflect increased DNA synthesisand cellular proliferation since high levels of AAH immunoreactivitywere observed in 100 percent of cholangiocarcinomas, but not in human orexperimental disease processes associated with regeneration ornonneoplastic proliferation of bile ducts. AAH overexpression andattendant high levels of beta hydroxylase activity lead to invasivegrowth of transformed neoplastic cells. Detection of an increase in HAAHexpression is useful for early and reliable diagnosis of the cancertypes which have now been characterized as overexpressing this geneproduct.

Diagnosis of Malignant Tumors

HAAH is overexpressed in many tumors of endodermal origin and in atleast 95% of CNS tumors compared to normal noncancerous cells. Anincrease in HAAH gene product in a patient-derived tissue sample (e.g.,solid tissue or bodily fluid) is carried out using standard methods,e.g., by Western blot assays or a quantitative assay such as ELISA. Forexample, a standard competitive ELISA format using an HAAH-specificantibody is used to quantify patient HAAH levels. Alternatively, asandwich ELISA using a first antibody as the capture antibody and asecond HAAH-specific antibody as a detection antibody is used.

Methods of detecting HAAH include contacting a component of a bodilyfluid with an HAAH-specific antibody bound to solid matrix, e.g.,microtiter plate, bead, dipstick. For example, the solid matrix isdipped into a patient-derived sample of a bodily fluid, washed, and thesolid matrix is contacted with a reagent to detect the presence ofimmune complexes present on the solid matrix.

Proteins in a test sample are immobilized on (bound to) a solid matrix.Methods and means for covalently or noncovalently binding proteins tosolid matrices are known in the art. The nature of the solid surface mayvary depending upon the assay format. For assays carried out inmicrotiter wells, the solid surface is the wall of the well or cup. Forassays using beads, the solid surface is the surface of the bead. Inassays using a dipstick (i.e., a solid body made from a porous orfibrous material such as fabric or paper) the surface is the surface ofthe material from which the dipstick is made. Examples of useful solidsupports include nitrocellulose (e.g., in membrane or microtiter wellform), polyvinyl chloride (e.g., in sheets or microtiter wells),polystyrene latex (e.g., in beads or microtiter plates, polyvinylidinefluoride (known as IMMULON™), diazotized paper, nylon membranes,activated beads, and Protein A beads. The solid support containing theantibody is typically washed after contacting it with the test sample,and prior to detection of bound immune complexes. Incubation of theantibody with the test sample is followed by detection of immunecomplexes by a detectable label. For example, the label is enzymatic,fluorescent, chemiluminescent, radioactive, or a dye. Assays whichamplify the signals from the immune complex are also known in the art,e.g., assays which utilize biotin and avidin.

An HAAH-detection reagent, e.g., an antibody, is packaged in the form ofa kit, which contains one or more HAAH-specific antibodies, controlformulations (positive and/or negative), and/or a detectable label. Theassay may be in the form of a standard two-antibody sandwich assayformat known in the art.

Production of HAAH-specific Antibodies

Anti-HAAH antibodies were obtained by techniques well known in the art.Such antibodies are polyclonal or monoclonal. Polyclonal antibodies wereobtained, for example, by the methods described in Ghose et al., Methodsin Enzymology, Vol. 93, 326-327, 1983. An HAAH polypeptide, or anantigenic fragment thereof, was used as the immunogen to stimulate theproduction of polyclonal antibodies in the antisera of rabbits, goats,sheep, or rodents. Antigenic polypeptides for production of bothpolyclonal and monoclonal antibodies useful as immunogens includepolypeptides which contain an HAAH catalytic domain. For example, theimmunogenic polypeptide is the full-length mature HAAH protein or anHAAH fragment containing the carboxyterminal catalytic domain e.g., anHAAH polypeptide containing the His motif of SEQ ID NO:2.

Antibodies which bind to the same epitopes as those antibodies disclosedherein as identified using standard methods, e.g., competitive bindingassays, known in the art.

Monoclonal antibodies were obtained by standard techniques. Ten μg ofpurified recombinant HAAH polypeptide was administered to miceintraperitoneally in complete Freund's adjuvant, followed by a singleboost intravenously (into the tail vein) 3-5 months after the initialinoculation. Antibody-producing hybridomas were made using standardmethods. To identify those hybridomas producing antibodies that arehighly specific for an HAAH polypeptide, hybridomas were screened usingthe same polypeptide immunogen used to immunize. Those antibodies whichwere identified as having HAAH-binding activity are also screened forthe ability to inhibit HAAH catalytic activity using the enzymaticassays described below. Preferably, the antibody has a binding affinityof at least about 10⁸ liters/mole and more preferably, an affinity of atleast about 10⁹ liters/mole.

Monoclonal antibodies are humanized by methods known in the art, e.g,MAbs with a desired binding specificity can be commercially humanized(Scotgene, Scotland; Oxford Molecular, Palo Alto, Calif.).

HAAH-specific intrabodies are produced as follows. Followingidentification of a hybridoma producing a suitable monoclonal antibody,DNA encoding the antibody is cloned. DNA encoding a single chainHAAH-specific antibody in which heavy and light chain variable domainsare separated by a flexible linker peptide is cloned into an expressionvector using known methods (e.g., Marasco et al., 1993, Proc. Natl.Acad. Sci. USA 90:7889-7893 and Marasco et al., 1997, Gene Therapy4:11-15). Such constructs are introduced into cells, e.g., usingstandard gene delivery techniques for intracellular production of theantibodies. Intracellular antibodies, i.e., intrabodies, are used toinhibit signal transduction by HAAH. Intrabodies which bind to acarboxyterminal catalytic domain of HAAH inhibit the ability of HAAH tohydroxylate EGF-like target sequences.

Methods of linking HAAH-specific antibodies (or fragments thereof) whichbind to cell surface exposed epitopes of HAAH on the surface of a tumorcell are linked to known cytotoxic agents, e.g, ricin or diptheriatoxin, using known methods.

Deposit of Biological Materials

Under the terms of the Budapest Treaty on the International Recognitionof the Deposit of Microorganisms for the Purpose of Patent Procedure,hybridoma FB501 (which produces monoclonal antibody FB50; designatedATCC accession no. PTA 3386), hybridoma HA386A (which producesmonoclonal antibody 86A; designated ATCC accession no. 3385), hybridomaHA15C7A (which produces monoclonal antibody 5C7; designated ATCCaccession no. 3383), and hybridoma HA219B (which produces monoclonalantibody 19B; designated ATCC accession no. 3384) were deposited on May17, 2001, with the American Type Culture Collection (ATCC) of 10801University Boulevard, Manassas, Va. 20110-2209 USA.

Applicants' assignee represents that the ATCC is a depository affordingpermanence of the deposit and ready accessibility thereto by the publicif a patent is granted. All restrictions on the availability to thepublic of the material so deposited will be irrevocably removed upon thegranting of a patent. The material will be available during the pendencyof the patent application to one determined by the Commissioner to beentitled thereto under 37 CFR 1.14 and 35 U.S.C. 122. The depositedmaterial will be maintained with all the care necessary to keep itviable and uncontaminated for a period of at least five years after themost recent request for the furnishing of a sample of the depositedplasmid, and in any case, for a period of at least thirty (30) yearsafter the date of deposit or for the enforceable life of the patent,whichever period is longer.

Applicant's assignee acknowledges its duty to replace the deposit shouldthe depository be unable to furnish a sample when requested due to thecondition of the deposit.

Methods of Treating Malignant Tumors

Patients with tumors characterized as overexpressing HAAH as such tumorsof endodermal origin or CNS tumors are treated by administering HAAHantisense nucleic acids.

Antisense therapy is used to inhibit expression of HAAH in patientssuffering from hepatocellular carcinomas, cholangiocarcinomas,glioblastomas and neuroblastomas. For example, an HAAH antisense strand(either RNA or DNA) is directly introduced into the cells in a form thatis capable of binding to the mRNA transcripts. Alternatively, a vectorcontaining a sequence which, which once within the target cells, istranscribed into the appropriate antisense mRNA, may be administered.Antisense nucleic acids which hybridize to target mRNA decrease orinhibit production of the polypeptide product encoded by a gene byassociating with the normally single-stranded mRNA transcript, therebyinterfering with translation and thus, expression of the protein. Forexample, DNA containing a promoter, e.g., a tissue-specific or tumorspecific promoter, is operably linked to a DNA sequence (an antisensetemplate), which is transcribed into an antisense RNA. By “operablylinked” is meant that a coding sequence and a regulatory sequence(s)(i.e., a promoter) are connected in such a way as to permit geneexpression when the appropriate molecules (e.g., transcriptionalactivator proteins) are bound to the regulatory sequence(s).

Oligonucleotides complementary to various portions of HAAH mRNA aretested in vitro for their ability to decrease production of HAAH intumor cells (e.g., using the FOCUS hepatocellular carcinoma (HCC) cellline) according to standard methods. A reduction in HAAH gene product incells contacted with the candidate antisense composition compared tocells cultured in the absence of the candidate composition is detectedusing HAAH-specific antibodies or other detection strategies. Sequenceswhich decrease production of HAAH in in vitro cell-based or cell-freeassays are then be tested in vivo in rats or mice to confirm decreasedHAAH production in animals with malignant neoplasms.

Antisense therapy is carried out by administering to a patient anantisense nucleic acid by standard vectors and/or gene delivery systems.Suitable gene delivery systems may include liposomes, receptor-mediateddelivery systems, naked DNA, and viral vectors such as herpes viruses,retroviruses, adenoviruses and adeno-associated viruses, among others. Areduction in HAAH production results in a decrease in signaltransduction via the IRS signal transduction pathway. A therapeuticnucleic acid composition is formulated in a pharmaceutically acceptablecarrier. The therapeutic composition may also include a gene deliverysystem as described above. Pharmaceutically acceptable carriers arebiologically compatible vehicles which are suitable for administrationto an animal: e.g., physiological saline. A therapeutically effectiveamount of a compound is an amount which is capable of producing amedically desirable result such as reduced production of an HAAH geneproduct or a reduction in tumor growth in a treated animal.

Parenteral administration, such as intravenous, subcutaneous,intramuscular, and intraperitoneal delivery routes, may be used todeliver nucleic acids or HAAH-inhibitory peptides or non-peptidecompounds. For treatment of CNS tumors, direct infusion intocerebrospinal fluid is useful. The blood-brain barrier may becompromised in cancer patients, allowing systemically administered drugsto pass through the barrier into the CNS. Liposome formulations oftherapeutic compounds may also facilitate passage across the blood-brainbarrier.

Dosages for any one patient depends upon many factors, including thepatient's size, body surface area, age, the particular nucleic acid tobe administered, sex, time and route of administration, general health,and other drugs being administered concurrently. Dosage for intravenousadministration of nucleic acids is from approximately 10⁶ to 10²² copiesof the nucleic acid molecule.

Ribozyme therapy is also be used to inhibit HAAH gene expression incancer patients. Ribozymes bind to specific mRNA and then cut it at apredetermined cleavage point, thereby destroying the transcript. TheseRNA molecules are used to inhibit expression of the HAAH gene accordingto methods known in the art (Sullivan et al., 1994, J. Invest. Derm.103:85S-89S; Czubayko et al., 1994, J. Biol. Chem. 269:21358-21363;Mahieu et al, 1994, Blood 84:3758-65; Kobayashi et al. 1994, Cancer Res.54:1271-1275).

Methods of Identifying Compounds that Inhibit HAAH Enzymatic Activity

Aspartyl (asparaginyl) beta-hydroxylaseydroxylase (AAH) activity ismeasured in vitro or in vivo. For example, HAAH catalyzesposttranslational modification of β carbon of aspartyl and asparaginylresidues of EGF-like polypeptide domains. An assay to identify compoundswhich inhibit hydroxylase activity is carried out by comparing the levelof hydroxylation in an enzymatic reaction in which the candidatecompound is present compared to a parallel reaction in the absence ofthe compound (or a predetermined control value). Standard hydroxylaseassays carried out in a testtube are known in the art, e.g., Lavaissiereet al., 1996, J. Clin. Invest. 98:1313-1323; Jia et al., 1992, J. Biol.Chem. 267:14322-14327; Wang et al., 1991, J. Biol. Chem.266:14004-14010; or Gronke et al., 1990, J. Biol. Chem. 265:8558-8565.Hydroxylase activity is also measured using carbon dioxide (¹⁴CO₂capture assay) in a 96-well microtiter plate format (Zhang et al., 1999,Anal. Biochem. 271:137-142. These assays are readily automated andsuitable for high throughput screening of candidate compounds toidentify those with hydroxylase inhibitory activity.

Candidate compound which inhibit HAAH activation of NOTCH are identifiedby detecting a reduction in activated NOTCH in a cell which expresses oroverexpresses HAAH, e.g., FOCUS HCC cells. The cells are cultured in thepresence of a candidate compound. Parallel cultures are incubated in theabsence of the candidate compound. To evaluate whether the compoundinhibits HAAH activation of NOTCH, translocation of activated NOTCH tothe nucleus of the cell is measured. Translocation is measured bydetecting a 110 kDa activation fragment of NOTCH in the nucleus of thecell. The activation fragment is cleaved from the large (approximately300 kDa) transmembrane NOTCH protein upon activation. Methods ofmeasuring NOTCH translocation are known, e.g, those described by Song etal., 1999, Proc. Natl. Acad. Sci U.S.A. 96:6959-6963 or Capobianco etal., 1997, Mol. Cell Biol. 17:6265-6273. A decrease in translocation inthe presence of the candidate compound compared to that in the absenceof the compound indicates that the compound inhibits HAAH activation ofNOTCH, thereby inhibiting NOTCH-mediated signal transduction andproliferation of HAAH-overexpressing tumor cells.

Methods of screening for compounds which inhibit phosphorylation of IRSare carried out by incubating IRS-expressing cells in the presence andabsence of a candidate compound and evaluating the level of IRSphosphorylation in the cells. A decrease in phosphorylation in cellscultured in the presence of the compound compared to in the absence ofthe compound indicates that the compound inhibits IRS-1 phosphorylation,and as a result, growth of HAAH-overexpressing tumors. Alternatively,such compounds are identified in an in vitro phosphorylation assay knownin the art, e.g., one which measured phosphorylation of a syntheticsubstrate such as poly (Glu/Tyr).

EXAMPLE 1 Increased Expression of HAAH is Associated with MalignantTransformation

HAAH is a highly conserved enzyme that hydroxylates EGF-like domains intransformation associated proteins. The HAAH gene is overexpressed inhuman hepatocellular carcinomas and cholangiocarcinomas. HAAH geneexpression was found to be undetectable during bile duct proliferationin both human disease and rat models compared to cholangiocarcinoma.Overexpression of HAAH in NIH-3T3 cells was associated with generationof a malignant phenotype, and enzymatic activity was found to berequired for cellular transformation. The data described below indicatethat overexpression of HAAH is linked to cellular transformation ofbiliary epithelial cells.

To identify molecules that are specifically overexpressed in transformedmalignant cells of human hepatocyte origin, the FOCUS hepatocellularcarcinoma (HCC) cell line was used as an immunogen to generatemonoclonal antibodies (mAb) that specifically or preferentiallyrecognize proteins associated with the malignant phenotype. A lambdaGT11 cDNA expression library derived from HepG2 HCC cells was screened,and HAAH-specific mAb produced against the FOCUS cell line was found torecognize an epitope on a protein encoded by an HAAH cDNA. The HAAHenzyme was found to be upregulated in several different humantransformed cell lines and tumor tissues compared to adjacent humantissue counterparts. The overexpressed HAAH enzyme in different humanmalignant tissues was found to be catalytically active.

HAAH gene expression was examined in proliferating bile ducts and in NIH3T3 cells. Its role in the generation of the malignant phenotype wasmeasured by the formation of transformed foci, growth in soft agar as anindex of anchorage independent growth and tumor formation in nude mice.The role of enzymatic activity in the induction of transformed phenotypewas measured by using a cDNA construct with a mutation in the catalyticsite that abolished hydroxylase activity. The results indicated that anincrease in expression of HAAH gene is associated with malignanttransformation of bile ducts.

The following materials and methods were used to generate the datadescribed below.

Antibodies

The FB50 monoclonal antibody was generated by cellular immunization ofBalb/C mice with FOCUS HCC cells. A monoclonal anti-Dengue virusantibody was used as a non-relevant control. The HBOH2 monoclonalantibody was generated against a 52 kDa recombinant HAAH polypeptide andrecognizes the catalytic domain of beta-hydroxylase from mouse and humanproteins. Polyclonal anti-HAAH antibodies cross-react with rathydroxylase protein. Control antibody anti-Erk-1 was purchased fromSanta Cruz Biotechnology, Inc, CA. Sheep anti-mouse and donkeyanti-rabbit antisera labeled with horseradish peroxidase were obtainedfrom Amersham, Arlington Heights, Ill.

Constructs

The murine full length AAH construct (pNH376) and the site-directedmutation construct (pNH376-H660) with abolished catalytic activity werecloned into the eukaryotic expression vector pcDNA3 (Invitrogen Corp.,San Diego, Calif.). The full length human AAH was cloned intoprokaryotic expression vector PBC-SK+ (Stratagene, La Jolla, Calif.).The full length human AAH (GENBANK Accession No. S83325) was subclonedinto the EcoRI site of the pcDNA3 vector.

Animal Model of Bile Duct Proliferation

Rats were divided into 9 separate groups of 3 animals each except forgroup 9 which contained 5 rats. Group 1 was the non-surgical controlgroup, and group 2 was the sham-operated surgical control. The remaininggroups underwent common bile duct ligation to induce intrahepatic bileduct proliferation and were evaluated at 6, 12, 24, 48 hours and 4, 8and 16 days as shown in Table 3. Animals were asphyxiated with CO₂, andliver samples were taken from left lateral and median lobes, fixed in 2%paraformaldehyde and embedded in paraffin. Liver samples (5 μm) were cutand stained with hematoxylin and eosin to evaluate intrahepatic bileduct proliferation. Immunohistochemistry was performed with polyclonalanti-HAAH antibodies that cross-react with the rat protein to determinelevels of protein expression.

Bile Duct Proliferation Associated with Primary Sclerosing Cholangitis(PSC)

Liver biopsy samples were obtained from 7 individuals with PSC andassociated bile duct proliferation. These individuals were evaluatedaccording to standard gastroenterohepatological protocols. Patients were22-46 years of age and consisted of 4 males and 3 females. Four hadassociated inflammatory bowel disease (3 ulcerative colitis and 1Crohn's colitis). All patients underwent a radiological evaluationincluding abdominal ultrasonography and endoscopic retrogradecholangiopancreaticography to exclude the diagnosis of extrahepaticbiliary obstruction. Tissue sections were prepared from paraffinembedded blocks and were evaluated by hematoxylin and eosin staining forbile duct proliferation. Expression of HAAH was determined byimmunohistochemistry using an HAAH-specific monoclonal antibody such asFB50.

Immunohistochemistry

Liver tissue sections (5 μm) were deparaffinized in xylene andrehydrated in graded alcohol. Endogenous peroxidase activity wasquenched by a 30-minute treatment with 0.6% H₂O₂ in 60% methanol.Endogenous biotin was masked by incubation with avidin-biotin blockingsolutions (Vector Laboratories, Burlingame, Calif.). The FB50 mAb (forPSC samples) and polyclonal anti-HAAH-hydroxylase antibodies (for ratliver samples) were added to slides in a humidified chamber at 4°C.overnight. Immunohistochemical staining was performed using a standardavidin-biotin horseradish peroxidase complex (ABC) method usingVectastain Kits with diaminobenzidine (DAB) as the chromogen accordingto manufacturer's instructions (Vector Laboratories, Inc., Burlingame,Calif.). Tissue sections were counterstained with hematoxylin, followedby dehydration in ethanol. Sections were examined by a light microscopyfor bile duct proliferation and HAAH protein expression. Paraffinsections of cholangiocarcinoma and placenta were used as positivecontrols, and hepatosteatosis samples were used as a negative controls.To control for antibody binding specificity, adjacent sections wereimmunostained in the absence of a primary antibody, or usingnon-relevant antibody to Dengue virus. As a positive control for tissueimmunoreactivity, adjacent sections of all specimens were immunostainedwith monoclonal antibody to glyceraldehyde 3-phosphate dehydrogenase.

Western Blot Analysis

Cell lysates were prepared in a standard radioimmunoprecipitation assay(RIPA) buffer containing protease inhibitors. The total amount ofprotein in the lysates was determined by Bio-Rad calorimetric assay (BioRad, Hercules, Calif.) followed by 10% sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred toPVDF membranes, and subjected to Western blot analysis using FB50,HBOH2, anti-Erk-1 (used as an internal control for protein loading) asprimary, sheep anti-mouse and donkey anti-rabbit antisera labeled withhorseradish peroxidase as secondary antibodies. Antibody binding wasdetected with enhanced chemiluminescence reagents (SuperSignal, PierceChemical Company, Rockford, Ill.) and film autoradiography. The levelsof immunoreactivity were measured by volume densitometry using NIH Imagesoftware.

Enzymatic Activity Assay

AAH activity was measured in cell lysates using the first EGF-likedomain of bovine protein S as substrate where ¹⁴C-labeledα-ketogluterate hydroxylates the domain releasing ¹⁴C containing CO2according to standard methods, e.g., those described by Jia et al.,1992, J. Biol. Chem. 267:14322-14327; Wang et al., 1991, J. Biol. Chem.266:14004-14010; or Gronke et al., 1990, J. Biol. Chem. 265:8558-8565.Incubations were carried out at 37° C. for 30 min in a final volume of40 μl containing 48 μg of crude cell extract protein and 75 μM EGFsubstrate.

Cell Transfection Studies

The NIH-3T3 cells were cultured in Dulbecco's modified Eagle's medium(DMEM; Mediatech, Washington, D.C.) supplemented with 10%heat-inactivated fetal calf serum (FCS; Sigma Chemical Co., St.Louis,Mo.), 1% L-glutamine, 1% non-essential amino acids and 1%penicillin-streptomycin (GIBCO BRL, Life Technologies, Inc., GrandIsland, N.Y.). Subconfluent NIH-3T3 cells (3×10⁵ cells/60-mm dish) weretransfected with 10 μg of one of the following plasmids: 1)non-recombinant pcDNA3 vector (Invitrogen Corp., San Diego, Calif.) as anegative control; 2) pNH376-H660, the murine AAH cDNA that was mutatedin the catalytic domain and cloned into the pcDNA3 vector driven by aCMV promoter; 3) pNH376, the wild type murine AAH cDNA cloned into thepcDNA3 vector; 4) pCDHH, wild type human AAH cDNA cloned into the pcDNA3vector; or 5) pLNCX-UP1, a cDNA that encodes v-Src oncogene (positivecontrol). Cells were transfected using the calcium phosphatetransfection kit according to manufacturer's instructions (5 Prime—3Prime, Inc., Boulder, Colo.). Comparison of cellular transfectionefficiency was assessed with the various constructs. For this procedure,confluent plates obtained 48 hours after transfection were split andreseeded into 12 separate 6-cm dishes, and 6 of them were made to growin the presence of 400 μg/ml G-418 (GIBCO BRL, Life Technologies, Inc.,Grant Island, N.Y.) containing medium. The number of G-418 resistantfoci was determined at 14 days after transfection and used to correctfor any variability in transfection efficiency.

Transformation Assay

The NIH-3T3 cells were transfected with the various constructs andallowed to reach confluence after 48 hours as described above. Each 6 cmdish was split and seeded into 12 different 6 cm dishes. While 6 of themwere made to grow in the presence of G-418 to detect transfectionefficiency, the other six were grown in complete medium without G-418and with a medium change every 4th day. The number of transformed fociwere counted in these plates without G-418 and expressed as transformedfoci per μg transfected DNA.

Anchorage-independent Cell Growth Assay

A limiting dilution technique (0.15 cell/well of a flat bottom96-well-plate) was performed on transfectants grown in G-418 in order toisolate cell clones with different levels of HAAH activity as measuredby Western blot analysis and enzymatic assay of hydroxylase activity.Cloned cell lines (1.0×10⁴ cells) were suspended in complete mediumcontaining 0.4% low-melting agarose (SeaPlaque GTG Agarose; FMCBioproducts, Rockland, Me.) and laid over a bottom agar mixtureconsisting of complete medium with 0.53% low-melting agarose. Each clonewas assayed in triplicate. The clones were seeded under these conditionsand 10 days later the size (positive growth>0.1 mm in diameter) andnumber of foci were determined.

Tumorigenicity in Nude Mice

The same clones as assessed in the anchorage independent growth assaywere injected into nude mice and observed for tumor formation.Tumorigenicity was evaluated using 10 animals in each of 4 groups(Charles River Labs., Wilmington, Mass.). Group 1 received 1×10⁵ cellsstably transfected with mock DNA, Group 2-4 received 1×10⁷ cells ofclones stable transfected with pNH376 and expressing various levels ofmurine HAAH protein. Nude mice were kept under pathogen-free conditionsin a standard animal facility. Thirty days after tumor cell inoculation,the animals were sacrificed using isofluorane (Aerrane, Anaquest, N.J.)containing chambers and the tumors were carefully removed and weightdetermined.

Animal Model of Bile Duct Proliferation

Following ligation of the common bile duct, intrahepatic bile ductproliferation was evident at 48 hours. Tissue samples obtained 8 and 16days following common bile duct ligation revealed extensive bile ductproliferation as shown in Table 3. TABLE 3 Bile duct proliferation andHAAH expression at different intervals after common bile duct ligationSurgical Group Procedure Microscopy* Immunohistochemistry 1 no surgerynormal negative 2 sham surgery normal negative 3 6 hours post normalnegative ligation 4 12 hours post normal negative ligation 5 24 hourspost normal negative ligation 6 48 hours post minimal bile negativeligation duct prolif. 7 4 days post moderate bile negative ligation ductprolif. 8 8 days post extensive negative ligation bile duct prolif. 9 16days post extensive negative ligation bile duct prolif.*Investigation was performed under light microscopy following ahematoxylin and eosin staining.

Immunohistochemical staining failed to detect sence of HAAH inproliferating bile ducts at any time. Analysis of HAAH expression inbile ducts derived from sham surgical controls was also negative, whileall samples exhibited positive immunoreactivity with control antibodiesto glyceraldehyde 3-phosphate dehydrogenase. Thus, bile ductproliferation was not associated with increased HAAH expression in thisstandard animal model system.

HAAH Expression in PSC

The liver biopsy specimens from patients with PSC exhibited bile ductproliferation accompanied by periductal fibrosis and a mononuclearinflammatory cell infiltrate without evidence of dysplasia. Adjacentsections immunostained with the an HAAH-specific monoclonal antibody hadno detectable HAAH immunoreactivity in proliferating bile ducts. Incontrast, sections of cholangiocarcinoma that were immunostainedsimultaneously using the same antibody and detection reagents manifestedintense levels of HAAH immunoreactivity in nearly all tumor cells,whereas adjacent sections of the cholangiocarcinomas exhibited anegative immunostaining reaction with monoclonal antibody to Denguevirus. These findings indicate that HAAH expression was associated withmalignant transformation rather than non-cancerous cellularproliferation of intrahepatic bile ducts.

HAAH Associated Transformation of NIH-3T3 Cells

The transforming capability of the murine and human AAH genes, as wellas the murine AAH mutant construct without enzymatic activity werecompared to mock DNA (negative control) and v-Src transfected NIH-3T3cells (positive control). The transforming capability of murine AAH wasfound to be 2-3 times that of vector DNA control as shown in FIG. 1. Thetransforming capacity of the human gene was greater than that observedwith the murine AAH (32±1.5 versus 13±2.6 transformed foci,respectively). The murine and human AAH transfected cells formed largefoci, resembling those of v-Src transfected fibroblasts, compared to theoccasional much smaller foci observed in cells transfected with vectorDNA that displayed the contact inhibition of fibroblast cell lines.Parallel experiments performed using the mutant pNH376-H660 constructwithout enzymatic activity revealed no transforming activity. Thisfinding indicates that the enzymatic activity of HAAH is required forthe transforming activity exhibited by the HAAH gene.

Anchorage-independent Cell Growth Assay

After transient transfection with the murine AAH construct, severaldifferent transformed foci were isolated for dilutional cloningexperiments to establish stable transfected cell clones with differentlevels of HAAH gene expression. Nine different cloned cell lines wereselected for further study. The expression level of the HAAH protein wasdetermined by Western blot analysis. Clones 7 and 18 had a modestincrease in HAAH protein expression, yet formed large colonies in softagar (FIG. 2). Protein loading was equivalent in all lanes as shown byimmunoblotting of the same membranes with an anti-Erk-1 monoclonalantibody. The increased protein expression was associated with increasedenzymatic activity as shown in FIG. 3. The capability of these clones toexhibit anchorage independent cell growth in soft agar is presented inFIG. 3. All 3 clones with increased HAAH gene expression demonstratedanchorage independent cell growth compared to the mock DNA transfectedclone.

Tumor Formation in Nude Mice

The 3 clones with increased HAAH gene expression were evaluated for theability to form tumors in nude mice. Tumor size in the mouse given clone18 was compared to a mock DNA transfected clone. Clones 7, 16 and 18were highly transformed in this assay and produced large tumors with amean weight of 2.5, 0.9 and 1.5 grams, respectively (FIG. 4). These dataindicate that overexpression of HAAH contributes to induction andmaintenance of the malignant phenotype in vivo.

High Level HAAH Expression is Indicative of Malignancy

In order to determine if HAAH expression was associated with malignancyrather than increased cell turnover, two models of bile ductproliferation were studied. In the animal model, ligation of the commonbile duct induced extensive intrahepatic bile duct proliferation, yetthere was no evidence of HAAH gene expression under these experimentalconditions as shown in Table 3. Similarly, HAAH gene expression wasassessed in a human disease model associated with bile ductproliferation since PSC is an autoimmune liver disease associated withdestruction as well as proliferation of the intra and extrahepatic bileducts. PSC is premalignant disease, and a significant proportion ofaffected individuals will eventually develop cholangiocarcinoma.However, no evidence for increased HAAH gene expression in the presenceof extensive bile duct proliferation.

Having established that HAAH protein levels were elevated incholangiocarcinoma and not in normal or proliferating bile ducts, therole of HAAH in the generation of a malignant phenotype was studied. TheHAAH gene was transfected into NIH-3T3 cells and cellular changes, e.g.,increased formation of transformed foci, colony growth in soft agar andtumor formation in nude mice associated with malignant transformation,were evaluated. The full-length murine and human AAH genes were clonedinto expression constructs and transiently transfected into NIH-3T3cells. An increased number of transformed foci was detected in cellstransfected both with the murine and human AAH genes as compared to mockDNA transfected controls. The increased number of transformed foci,after controlling for transfection efficiency, was not as high comparedto v-Src gene transfected cells used as a positive control. Theenzymatic activity of the HAAH gene was required for a malignantphenotype because a mutant construct which abolished the catalytic sitehad no transforming properties. Several stable transfectants and clonedNIH-3T3 cell lines with a modest increase in HAAH protein levels andenzymatic activity were established. Such cell lines were placed in softagar to examine anchorage independent cell growth as another property ofthe malignant phenotype. All cell lines grew in soft agar compared tomock DNA transfected control, and there was a positive correlationbetween the cellular level of HAAH gene expression and the number andsize of colonies formed. Three of these cloned cell lines formed tumorsin nude mice. All three cell lines with increased HAAH expression wereoncogenic as shown by the development of large tumors as anotherwell-known characteristic of the transformed phenotype.

To determine whether cellular changes induced by overexpression of HAAHwere related to the enzymatic function, a site-directed mutation wasintroduced into the gene that changed the ferrous iron binding site fromhistidine to lysine at 660th position of mouse HAAH thereby abolishinghydroxylase activity of the murine HAAH. A corresponding mutation inHAAH is used as a dominant negative mutant to inhibit HAAH hydroxylaseactivity. The pNH376-H660 construct had no transformation activityindicating cellular changes of the malignant phenotype induced byoverexpression depends on the enzymatic activity of the protein.

Notch receptors and their ligands have several EGF-like domains in theN-terminal region that contain the putative consensus sequence forbeta-hydroxylation. Notch ligands are important elements of the Notchsignal transduction pathway and interaction of Notch with its ligandsoccurs by means of EGF-like domains of both molecules. Point mutationsaffecting aspartic acid or asparagine residues in EGF-like domains thatare the targets for beta-hydroxylation by HAAH reduce calcium bindingand protein-protein interactions involved in the activation ofdownstream signal transduction pathways. Overexpression of HAAH andNotch protein hydroxylation by HAAH contributes to malignancy. Tumorgrowth is inhibited by decreasing Notch protein hydroxylation by HAAH

The data presented herein is evidence that high-level HAAH expression islinked to malignant transformation. An increase in expression of theHAAH cDNA in NIH-3T3 cells induced a transformed phenotype manifested byincreased numbers of transformed foci, anchorage-independent growth, andtumorigenesis in nude mice. In addition, intact HAAH-enzyme was found tobe required for HAAH-associated transformation. Accordingly, inhibitionof as little as 20% of endogenous HAAH enzymatic activity or expressionconfers a therapeutic benefit. For example, clinical benefit is achievedby 50%-70% inhibition of HAAH expression or activity afteradministration of an HAAH inhibitory compound compared to the levelassociated with untreated cancer cell or a normal noncancerous cell.

HAAH is regulated at the level of transcription. Only modest increasesin HAAH expression and enzyme activity were required for cellulartransformation. These results indicate that increased HAAH geneexpression and enzyme activity contribute to the generation ormaintenance of the transformed phenotype and that decreasingtranscription of the HAAH gene or decreasing enzymatic activity of theHAAH gene product leads to a decrease in malignancy. Accordingly, HAAHtranscription is inhibited by administering compounds which decreasebinding of Fos and/or Jun (elements which regulate HAAH transcription)to HAAH promoter sequences.

Since HAAH is up-regulated with malignant transformation of bile ductepithelium, and HAAH immunoreactivity is detectable on tumor cellsurface membranes, HAAH is also a molecule to which to target acytotoxic agent, e.g., by linking the cytotoxic agent to a compound thatbinds to HAAH expressed on the surface of a tumor cell. Assay of HAAHprotein levels in either biological fluids such as bile, or cellsobtained by fine needle aspiration is a diagnostic marker of humancholangiocarcinoma.

EXAMPLE 2 Expression of AAH and Growth and Invasiveness of Malignant CNSNeoplasms

AAH is abundantly expressed in carcinomas and trophoblastic cells, butnot in most normal cells, including those of CNS origin. High levels ofAAH expression were observed in 15 of 16 glioblastomas, 8 of 9anaplastic oligodendrogliomas, and 12 of 12 primitive neuroectodermaltumors (PNETS). High levels of AAH immunoreactivity were primarilylocalized at the infiltrating edges rather than in the central portionsof tumors. Double-label immunohistochemical staining demonstrated areciprocal relationship between AAH and tenascin, a substrate for AAHenzyme activity. PNET2 neuronal cell lines treated with phorbol estermyristate or retinoic acid to stimulate neuritic extension and invasivegrowth exhibited high levels of AAH expression, whereas H₂O₂-inducedneurite retraction resulted in down-regulation of AAH. PNET2 neuronalcells that stably over-expressed the human AAH cDNA had increased levelsof PCNA and Bcl-2, and reduced levels of p21/Waf1 and p16, suggestingthat AAH overexpression results in enhanced pathological cellproliferation, cell cycle progression, and resistance to apoptosis. Inaddition, the reduced levels of p16 observed in AAH-transfectantsindicate that AAH over-expression confers enhanced invasive growth ofneoplastic cells since deletion or down-regulation of the p16 genecorrelates with more aggressive and invasive in vivo growth ofglioblastomas. Increased AAH immunoreactivity was detected at theinfiltrating margins of primary malignant CNS neoplasms, furtherindicating a role of HAAH in tumor invasiveness.

The following materials and methods were used to generate the datadescribed below.

Analysis of AAH Immunoreactivity in Primary Human Malignant CNSNeoplasms:

AAH immunoreactivity was examined in surgical resection specimens ofglioblastoma (N=16), anaplastic oligodendroglioma (N=9), and primitiveneuroectodermal tumor (PNET; supratentorial neuroblastomas (N=3) andmedulloblastomas (N=9). The histopathological sections were reviewed toconfirm the diagnoses using standard criteria. Paraffin sections fromblocks that contained representative samples of viable solid tumor, ortumor with adjacent intact tissue were studied. Sections from normaladult postmortem brains (N=4) were included as negative controls. AAHimmunoreactivity was detected using qn HAAH-specific monoclonalantibody. Immunoreactivity was revealed by the avidin-biotin horseradishperoxidase complex method (Vector ABC Elite Kit; Vector Laboratories,Burlingame, Calif.) using 3-3′ diaminobenzidine (DAB) as the chromogen(24) and hematoxylin as a counterstain.

Tenascin and laminin are likely substrates for AAH due to the presenceof EGF-like repeats within the molecules. Double-immunostaining studieswere performed to co-localize AAH with tenascin or laminin. The AAHimmunoreactivity was detected by the ABC method with DAB as thechromogen, and tenascin or laminin immunoreactivity was detected by theavidin-biotin alkaline phosphatase complex method (Vector Laboratories,Burlingame, Calif.) with BCIP/NBT as the substrate. As positive andnegative controls, adjacent sections were immunostained with monoclonalantibody to glial fibrillary acidic protein (GFAP) and Hepatitis Bsurface antigen. All specimens were batch immunostained using the sameantibody dilutions and immunodetection reagents.

Cell Lines and Culture Conditions

Studies were conducted to determine whether AAH expression was modulatedwith neurite (filopodia) extension (sprouting) as occurs with invasivegrowth of malignant neoplasms. Human PNET2 CNS-derived and SH-Sy5yneuroblastoma cells were cultured and stimulated for 0, 1, 2, 3, 5, or 7days with 100 nM phorbol 12-ester 13-acetate or 10 μM retinoic acid toinduce sprouting. In addition, to examine the effects of neuriteretraction on AAH expression, subconfluent cultures were treated for 24hours with low concentrations (10-40 μM) of H₂O₂. For both studies, AAHexpression was evaluated by Western blot analysis using the anHAAH-specific antibody.

Generation of PNET2 AAH-transfected Clones

The full-length human AAH cDNA (SEQ ID NO:3) was ligated into thepcDNA3.1 mammalian expression vector in which gene expression was underthe control of a CMV promoter (Invitrogen Corp., San Diego, Calif.).PNET2 cells were transfected with either PHAAH or pcDNA3 (negativecontrol) using Cellfectin reagent (Gibco BRL, Grand Island, N.Y.).Neomycin-resistant clones were selected for study if the constitutivelevels of AAH protein expression were increased by at least two-foldrelative to control (pcDNA3) as detected by Western blot analysis. Todetermine how AAH overexpression altered the expression of genes thatmodulate the transformed phenotype, the levels of proliferating cellnuclear antigen (PCNA), p53, p21/Waf1, Bcl-2, and p16 were measured incell lysates prepared from subconfluent cultures of AAH (N=5) and pcDNA3(N=5) stably transfected clones. PCNA was used as marker of cellproliferation. p53, p21/Waf1, and Bcl-2 levels were examined todetermine whether cells that over-expressed AAH were more prone to cellcycle progression and more resistant to apoptosis. The levels of p16were assessed to determine whether AAH over-expression has a role intumor invasiveness.

Western Blot Analysis

Cells grown in 10 cm² dishes were lysed and 5 homogenized in a standardradioimmunoprecipitation assay RIPA buffer containing protease andphosphatase inhibitors. The supernatants collected after centrifugingthe samples at 12,000×g for 10 minutes to remove insoluble debris wereused for Western blot analysis. Protein concentration was measured usingthe BCA assay (Pierce Chemical Co, Rockford, Ill.). Samples containing60 μg of protein were electrophoresed in sodium dodecyl sulfatepolyacrylamide gels (SDS-PAGE) and subjected to Western blot analysis.Replicate blots were probed with the individual antibodies.Immunoreactivity was detected with horseradish peroxidase conjugated IgG(Pierce Chemical Co, Rockford, Ill.) and enhanced chemiluminescencereagents. To quantify the levels of protein expression, non-saturatedautoradiographs were subjected to volume densitometry using NIH Imagesoftware, version 1.6. Statistical comparisons between pHAAH and pcDNA3transfected cells were made using Student T tests.

Antibodies

HAAH-specific monoclonal antibody generated against the FOCUShepatocellular carcinoma cells were used to detect AAH immunoreactivity.Monoclonal antibodies to tenascin, and glial fibrillary acidic protein,and rabbit polyclonal antibody to laminin were purchased from Sigma Co(St. Louis, Mo.). Rabbit polyclonal antibody to human p16 was purchasedfrom Santa Cruz Biotechnology Inc. (Santa Cruz, Calif.). The 5C3negative control monoclonal antibody to Hepatitis B surface antigen wasgenerated using recombinant protein and used as a negative control.

AAH Immunoreactivity in Primary Malignant Brains Tumors

AAH immunoreactivity was detected in 15 of 16 glioblastomas, 8 of 9anaplastic oligodendrogliomas, and all 12 PNETs. AAH immunoreactivitywas localized in the cytoplasm, nucleus, and cell processes. The tissuedistribution of AAH immunoreactivity was notable for the intenselabeling localized at the interfaces between tumor and intact brain, andthe conspicuously lower levels of immunoreactivity within the centralportions of the tumors. High levels of AAH immunoreactivity were alsoobserved in neoplastic cells distributed in the subpial zones,leptomeninges, Virchow-Robin perivascular spaces, and in individual orsmall clusters of neoplastic cells that infiltrated the parenchyma. Incontrast, AAH immunoreactivity was not detectable in normal brain. Thedistribution of AAH immunoreactivity appeared not to be strictlycorrelated with DNA synthesis since the density of nuclei in mitosis(1-5%) was similar in the central and peripheral portions of the tumors.

Relationship Between AAH and Tenascin Immunoreactivity in Glioblastomas

Tenascin is an extracellular matrix-associated antigen expressed inmalignant gliomas. Tenascin contains EGF-like domains within themolecule, a substrate for HAAH hydroxylation. To localize AAH inrelation to tenascin immunoreactivity in malignant brain tumors,double-label immunohistochemical staining was performed in which AAH wasdetected using a brown chromogen (DAB), and tenascin, a blue chromogen(BCIP/NBT). Adjacent sections were similarly double-labeled toco-localize AAH with laminin, another EGF domain containingextracellular matrix molecule expressed in the CNS. Intense levels oftenascin immunoreactivity were observed in perivascular connectivetissue and in association with glomeruloid proliferation of endothelialcells. The double-labeling studies demonstrated a reciprocalrelationship between AAH and tenascin immunoreactivity such that highlevels of AAH were associated with low or undetectable tenascin, and lowlevels of AAH were associated with abundant tenascin immunoreactivity.Although laminins are also likely substrates for AAH enzyme activity dueto the EGF repeats within the molecules, double labeling studiesrevealed only low levels of laminin immunoreactivity throughout thetumors and at interfaces between tumor and intact tissue.

Analysis of AAH Expression in Neuronal Cell Lines Treated with PMA or RA

Neuritic sprouting/filopodia extension marks invasive growth ofneoplastic neuronal cells. PMA activates protein kinase C signaltransduction pathways that are involved in neuritic sprouting. Retinoicacid binds to its own receptor and the ligand-receptor complextranslocates to the nucleus where it binds to specific consensussequences present in the promoter/enhancer regions of target genesinvolved in neuritic growth. Both PNET2 and SH-Sy5y cells can be inducedto sprout by treatment with PMA (60-120 nM) or retinoic acid (5-10 μM).FIGS. 5A-D depict data from representative Western blot autoradiographs;the bar graphs correspond to the mean ±S.D. of results obtained fromthree experiments. Western blot analysis with the FB50 antibody detecteddoublet bands corresponding to protein with an molecular mass ofapproximately 85 kDa. Untreated PNET2 cells had relatively low levels ofAAH immunoreactivity (FIG. 5A), whereas untreated SH-Sy5y cells hadreadily detected AAH expression (FIG. 5B). Untreated PNET2 cellsexhibited polygonal morphology with coarse, short radial cell processes,whereas SH-Sy5y cells were slightly elongated and spontaneously extendfine tapered processes. Both cell lines manifested time-dependentincreases in the levels of AAH immunoreactivity following either RA(FIGS. 5A and 5B) or PMA (FIG. 5C) stimulation and neurite extension. InPNET2 cells, the levels of AAH protein increased by at least two-fold 24hours after exposure to RA or PMA, and high levels of AAH were sustainedthroughout the 7 days of study. In SH-Sy5y cells, the RA- orPMA-stimulated increases in AAH expression occurred more gradually andwere highest after 7 days of treatment (FIG. 5B).

To examine the effect of AAH expression on neurite retraction, PNET2 andSH-Sy5y cells were treated with low concentrations (8-40 μM) of H₂O₂.After 24 hours exposure to up to 40 μM H₂O₂, although most cellsremained viable (Trypan blue dye exclusion), they exhibited neuriteretraction and rounding. Western blot analysis using the FB50 antibodydemonstrated H2O2 dose-dependent reductions in the levels of AAH protein(FIG. 5D).

Effects of AAH Over-expression in PNET2 Cells

To directly assess the role of AAH overexpression in relation to themalignant phenotype, PNET2 cells were stably transfected with the humanfull-length CDNA with gene expression under control of a CMV promoter(pHAAH). Neomycin-resistant clones that had at least two-fold higherlevels of AAH immunoreactivity relative to neomycin-resistant pcDNA3(mock) clones were studied. Since aggressive behavior of malignantneoplasms is associated with increased DNA synthesis, cell cycleprogression, resistance to apoptosis, and invasive growth, the changesin phenotype associated with constitutive over-expression of AAH werecharacterized in relation to PCNA, p21/Waf1, p53, Bcl-2, and p16. PCNAwas used as an index of DNA synthesis and cell proliferation. p21/Waf1is a cell cycle inhibitor. Expression of the p53 tumor-suppressor geneincreases prior to apoptosis, whereas bcl-2 inhibits apoptosis andenhances survival of neuronal cells. p16 is an oncosuppressor gene thatis often either down-regulated or mutated in infiltrating malignantneoplasms.

Five pHAAH and 5 pcDNA3 clones were studied. Increased levels of AAHexpression in the PHAAH transfected clones was confirmed by Western(FIG. 6) and Northern blot analyses. Western blot analysis using celllysates from cultures that were 70 to 80 percent confluent demonstratedthat constitutively increased levels of AAH expression (approximately 85kDa; P<0.05) in pHAAH-transfected cells were associated withsignificantly increased levels of PCNA (approximately 35 kDa; P<0.01)and Bcl-2 (approximately 25 kDa; P<0.05), and reduced levels of p21/Waf1(approximately 21 kDa; P<0.001) and p16 (approximately 16 kDa; P<0.001)(FIG. 6). However, the pHAAH stable transfectants also exhibited higherlevels of wild-type p53 (approximately 53-55 kDa). Although AAHexpression (85 kDa protein) in the stable transfectants was increased byonly 75 to 100 percent, the levels of p16 and p21/Waf1 were sharplyreduced, and PCNA increased by nearly two-fold (FIG. 6).

Increased AAH Expression is Indicative of Growth and Invasiveness ofMalignant CNS Neoplasms

The data described herein demonstrates that AAH overexpression is adiagnostic tool by which to identify primary malignant CNS neoplasms ofboth neuronal and glial cell origin. Immunohistochemical stainingstudies demonstrated that AAH overexpression was detectable mainly atthe interfaces between solid tumor and normal tissue, and ininfiltrating neoplastic cells distributed in the subpial zones,leptomeninges, perivascular spaces, and parenchyma. In vitro experimentsdemonstrated that AAH gene expression was modulated with neurite(filopodium) extension and invasiveness and down-regulated with neuriteretraction. In addition, PNET2 cells stably transfected with the AAHcDNA exhibited increased PCNA and bcl-2, and reduced Waf1/p21 and p16expression. Therefore, AAH overexpression contributes to the transformedphenotype of CNS cells by modulating the expression of other genes thatpromote cellular proliferation and cell cycle progression, inhibitapoptosis, or enhance tumor cell invasiveness.

The data demonstrated readily detectable AAH mRNA transcripts (4.3 kBand 2.6 kB) and proteins (85 kDa and 50-56 kDa) in PNET2 and SH-Sy5ycells, but not in normal brain. Correspondingly, high levels of AAHimmunoreactivity were observed in 35 of the 37 in malignant primaryCNS-derived neoplasms studied, whereas the 4 normal control brains hadno detectable AAH immunoreactivity. The presence of high-level AAHimmunoreactivity at the infiltrating margins and generally not in thecentral portions of the tumors indicates that AAH overexpression isinvolved in the invasive growth of CNS neoplasms. Administration ofcompounds which decrease AAH expression or enzymatic activity inhibitsproliferation of CNS tumors which overexpress AAH, as well as metastasesof CNS tumors to other tissue types.

The AAH enzyme hydroxylates EGF domains of a number of proteins.Tenascin, an extracellular matrix molecule that is abundantly expressedin malignant gliomas, contains EGF-like domains. Since tenascin promotestumor cell invasion, its abundant expression in glioblastomas representsan autocrine mechanism of enhanced tumor cell growth vis-á-vis thefrequent overexpression of EGF or EGF-like receptors in malignant glialcell neoplasms. Analysis of the functional domains of tenascinsindicated that the mitogenic effects of this family of molecules arelargely mediated by the fibronectin domains, and that the EGF-likedomains inhibit growth, cell process elongation, and matrix invasion.Therefore, hydroxylation of the EGF-like domains by AAH represents animportant regulatory factor in tumor cell invasiveness.

Double-label immunohistochemical staining studies demonstrated areciprocal relationship between AAH and tenascin immunoreactivity suchthat high levels AAH immunoreactivity present at the margins of tumorswere associated with low levels of tenascin, and low levels of AAH wereoften associated with high levels of tenascin. These observationsindicated that AAH hydroxylation of EGF-like domains of tenascin altersthe immunoreactivity of tenascin protein, and in so doing, facilitatesthe invasive growth of malignant CNS neoplasms into adjacent normaltissue and perivascular spaces.

AAH immunoreactivity was examined in PNET2 and SH-Sy5y neuronal cellsinduced to undergo neurite extension with PMA or retinoic acid, orneurite retraction by exposure to low doses of H₂O₂. AAH expression wassharply increased by PMA- or retinoic acid-induced neurite (filopodium)extension, and inhibited by H2O2-induced neurite retraction and cellrounding. Neurite or filopodium extension and attachment toextracellular matrix are required for tumor cell invasion in the CNS.The EGF-like domains of tenascin inhibit neuritic and glial cell growthinto the matrix during development.

To directly examine the role of AAH overexpression in relation to thetransformed phenotype, genes modulated with DNA synthesis, cell cycleprogression, apoptosis, and tumor invasiveness were examined in neuronalcell clones that stably over-expressed the human AAH cDNA. The findingsof increased PCNA and reduced Waf1/p21 immunoreactivity indicated thatAAH overexpression enhances cellular proliferation and cell cycleprogression. In addition, the finding of increased Bcl-2 expressionindicated that AAH overexpression contributes to the transformedphenotype by increasing cellular resistance to apoptosis. The apparentlycontradictory finding of higher levels of p53 in the cells thatoverexpressed AAH is explained by the observation that high levels ofwildtype p53 in immature neuronal cells were associated with neuriticgrowth (invasiveness) rather than apoptosis. Levels of p16 were reduced(compared to normal cells) or virtually undetectable in cells thatconstitutively overexpressed AAH; a deletion mutation of the p16 genehas been correlated with invasive growth and more rapid progression ofmalignant neoplasms, including those of CNS origin. These data indicatethat p16 expression is modulated by AAH.

EXAMPLE 3 Increased HAAH Production and IRS-mediated Signal Transduction

IRS-1 mediated signal transduction pathway is activated in 95% of humanHCC tumors compared to the adjacent uninvolved liver tissue. HAAH is adownstream effector gene involved in this signal transduction pathway.HAAH gene upregulation is closely associated with overexpression ofIRS-1 in HCC tumors as revealed by immunohistochemical staining andWestern blot analysis. A high level of HAAH protein is expressed in HCCand cholangiocarcinoma compared to normal hepatocytes and bile ducts.Both of these tumors also exhibit high level expression of IRS-1 byimmunohistochemical staining. FOCUS HCC cell clones stably transfectedwith a C-terminal truncated dominant negative mutant of IRS-1, whichblocks insulin and IGF-1 stimulated signal transduction, was associatedwith a striking reduction in HAAH gene expression in liver. In contrast,transgenic mice overexpressing IRS-1 demonstrate an increase in HAAHgene expression by Western blot analysis. Insulin stimulation of FOCUSHCC cells (20 and 40 U) in serum free medium and after 16 hr of serumstarvation demonstrated upregulation of HAAH gene expression. These dataindicate that HAAH gene expression is a downstream effector of the IRS-1signal transduction pathway.

EXAMPLE 4 Effects of HAAH Expression Levels on the Characteristics ofthe Malignant Phenotype

Overexpression of IRS-1 in NIH 3T3 cells induces transformation. Thefull-length murine HAAH construct was cloned into the pcDNA3 eukaryoticexpression vector. A second murine construct encoded HAAH with abolishedcatalytic activity due to a site directed mutation. The full-lengthhuman HAAH cDNA was cloned into the pcDNA3 expression vector as well asa plasmid that encodes v-src which was used as a positive control fortransformation activity. Standard methods were used for transfection ofNIH 3T3 cells, control for transfection efficiency, assays of HAAHenzymatic activity, transformation by analysis of foci formation,anchorage-independent cell growth assays and analysis of tumorigenicityin nude mice. The data indicate that HAAH overexpression is associatedwith generation of a malignant phenotype. TABLE 4 Overexpression ofenzymatically active HAAH indicates malignancy # of cDNA # of foci ±S.D.^(b) NIH 3T3 clone colonies^(e) pcDNA3  6.0 ± 3.3 pcDNA 0.4 ± 0.5(mock) (mock) murine 14.0 ± 2.9 clone 18^(d) 6.2 ± 2.9 HAAH mutantmurine  1.6 ± 1.0 clone 16^(e) 4.7 ± 6.5 HAAH^(a) human 32.0 ± 5.4 HAAHv-scr 98.0 ± 7.1^(a)enzymatically inactive HAAH^(b)P < 0.01 compared to mock and mutant murine HAAH^(c)P < 0.001 compared to mock^(d)_Clone 18 is a stable cloned NIH 3T3 cell line that overexpressionhuman HAAH by approximately two fold.^(e)Clone 16 is a stable cloned NIH 3T3 cell line that overexpresseshuman HAAH by about 50%.

These data indicate that overexpression of HAAH is associated withformation of transformed foci. Enzymatic activity is required forcellular transformation to occur. Cloned NIH 3T3 cell lines withincreased human HAAH gene expression grew as solid tumors in nude mice.HAAH is a downstream effector gene of the IRS-1 signal transductionpathway.

EXAMPLE 5 Inhibition of HAAH Gene Expression

The FOCUS HCC cell line from which the human HAAH gene was initiallycloned has a level of HAAH expression that is approximately 3-4 foldhigher than that found in normal liver. To make an HAAH antisenseconstruct, the full length human HAAH cDNA was inserted in the oppositeorientation into a retroviral vector containing a G418 resistant gene,and antisense RNA was produced in the cells. Shorter HAAH antisensenucleic acids, e.g., those corresponding to exon 1 of the HAAH gene arealso used to inhibit HAAH expression.

FOCUS cells were infected with this vector and the level of HAAH wasdetermined by Western blot analysis. A reduction in HAAH gene expressionwas observed. Growth rate and morphologic appearance of cells infectedwith a retrovirus containing a nonrelevant Green Fluorescent Protein(GFP) also inserted in the opposite orientation as a control (FIG. 8).Cells (harboring the HAAH antisense construct) exhibited a substantialchange in morphology characterized by an increase in the cytoplasm tonuclear ratio as well as assuming cell shape changes that werereminiscent of normal adult hepatocytes in culture. Cells with reducedHAAH levels grew at a substantially slower rate than retroviral infectedcells expressing antisense (GFP) (control) as shown in FIG. 8. Areduction in HAAH gene expression was associated with a moredifferentiated noncancerous “hepatocyte like” phenotype. Expression ofHAAH antisense sequences are used to inhibit tumor growth rate.Reduction of HAAH cellular levels results in a phenotype characterizedby reduced formation of transformed foci, low level or absent anchorageindependent growth in soft agar, morphologic features of differentiatedhepatocytes as determined by light and phase contrast microscopy, and notumor formation (as tested by inoculating the cells into nude mice).

EXAMPLE 6 Human IRS-1 Mutants

Insulin/IGF-1 stimulated expression of HAAH in HCC cell lines.Dominant-negative IRS-1 cDNAs mutated in the plextrin andphosphotryosine (PTB) domains, and Grb2, Syp and PI3K binding motifslocated in the C-terminus of the molecule were constructed. Human IRS-1mutant constructs were generated to evaluate how HAAH gene expression isupregulated by activation of the IRS-1 growth factor signal transductioncascade. Specific mutations in the C terminus of the hIRS-1 moleculeabolished the various domains which bind to SH2-effector proteins suchas Grb2, Syp and PI3K. The human IRS-1 protein contains the same Grb2and Syp binding motifs of 897YVNI (underlined in Table 5, below and1180YIDL (underlined in Table 5, below), respectively, as the rat IRS-1protein. Mutants of hIRS-1 were constructed by substitution of a TATcodon (tyrosine) with a TTT codon (phenylalanine), in these motifs byuse of oligonucleotide-directed mutagenesis suing the following primers:(5′-GGGGGAATTTGTCAATA-3′ (SEQ ID NO:8) and 5′-GAATTTGTTAATATTG-3′ (SEQID NO:9), respectively). The cDNAs of hIRS-1 (wild-type) and mutants(tyrosine 897-to-phenylalanine and tyrosine 1180-to-phenylalanine) weresubcloned into the PBK-CMV expression vector and designated ashIRS-1-wt, 897F, Δ-Grb2), 1180F, and ΔSyp. TABLE 5 Human IRS-1 aminoacid sequence MASPPESDGF SDVRKVGYLR KPKSMHKRFF VLRAASEAGG 61 PARLEYYENEKKWRHKSSAP KRSIPLESCF NINKRADSKN KHLVALYTRD EHFAIAADSE 121 AEQDSWYQALLQLHNRAKGH HDGAAALGAG GGGGSCSGSS GLGEAGEDLS YGDVPPGPAF 181 KEVWQVILKPKGLGQTKNLI GIYRLCLTSK TISFVKLNSE AAAVVLQLMN IRRCGHSENF 241 FFIEVGRSAVTGPGEFWMQV DDSVVAQNNH ETILEANRAM SDEFRPRSKS QSSSNCSNPI 301 SVPLRRHHLNNPPPSQVGLT RRSRTESITA TSPASMVGGK PGSFRVRASS DGEGTMSRPA 361 SVDGSPVSPSTNRTHAHRHR GSARLHPPLN HSRSIPMPAS RCSPSATSPV SLSSSSTSGH 421 GSTSDCLFPRRSSASVSGSP SDGGFISSDE YGSSPCDFRS SFRSVTPDSL GHTPPARGEE 481 ELSNYICMGGKGPSTLTAPN GHYILSRGGN GHRCTPGTGL GTSPALAGDE AASAADLDNR 541 FRKRTHSAGTSPTITHQKTP SQSSVASIEE YTEMMPAYPP GGGSGGRLPG HRHSAFVPTR 601 SYPEEGLEMHPLERRGGHHR PDSSTLHTDD GYMPMSPGVA PVPSGRKGSG DYMPMSPKSV 661 SAPQQIINPIRRHPQRVDPN GYMMMSPSGG CSPDIGGGPS SSSSSSNAVP SGTSYGKLWT 721 NGVGGHHSHVLPHPKPPVES SGGKLLPCTG DYMNNSPVGD SNTSSPSDCY YGPEDPQHKP 781 VLSYYSLPRSFKHTQRPGEP EEGARHQHLR LSTSSGRLLY AATADDSSSS TSSDSLGGGY 841 CGARLEPSLPHPHHQVLQPH LPRKVDTAAQ TNSRLARPTR LSLGDPKAST LPRAREQQQQ 901 QQPLLHPPEPKSPGEYVNIE FGSDQSGYLS GPVAFHSSPS VRCPSQLQPA PREEETGTEE 961 YMKMDLGPGRRAAWQESTGV EMGRLGPAPP GAASICRPTR AVPSSRGDYM TMQMSCPRQS 1021 YVDTSPAAPVSYADMRTGIA AEEVSLPPAT MAAASSSSAA SASPTGPQGA AELAAHSSLL 1081 GGPQGPGGMSAFTRVNLSPN RNQSAKVIRA DPQGCRRRHS SETFSSTPSA TRVGNTVPFG 1141 AGAAVGGGGGSSSSSEDVKR HSSASFENVW LRPGELGGAP KEPAKLCGAA GGLENGLNYI 1201 DLDLVKDFKQCPQECTPEPQ PPPPPPPHQP LGSGESSSTR RSSEDLSAYA SISFQKQPED RQ(SEQ ID NO:5; GENBANK Accession No. JS0670; pleckstrin domain spansresidues 11-113, inclusive; Phosphate-binding residues include 46, 465,551, 612, 632, 662, 732, 941, 989, or 1012 of SEQ ID NO:5)

TABLE 6 Human IRS-1 cDNA cggcggcgcg gtcggagggg gccggcgcgc agagccagac 61gccgccgctt gttttggttg gggctctcgg caactctccg aggaggagga ggaggaggga 121ggaggggaga agtaactgca gcggcagcgc cctcccgagg aacaggcgtc ttccccgaac 181ccttcccaaa cctcccccat cccctctcgc ccttgtcccc tcccctcctc cccagccgcc 241tggagcgagg ggcagggatg agtctgtccc tccggccggt ccccagctgc agtggctgcc 301cggtatcgtt tcgcatggaa aagccacttt ctccacccgc cgagatgggc ccggatgggg 361ctgcagagga cgcgcccgcg ggcggcggca gcagcagcag cagcagcagc agcaacagca 421acagccgcag cgccgcggtc tctgcgactg agctggtatt tgggcggctg gtggcggctg 481ggacggttgg ggggtgggag gaggcgaagg aggagggaga accccgtgca acgttgggac 541ttggcaaccc gcctccccct gcccaaggat atttaatttg cctcgggaat cgctgcttcc 601agaggggaac tcaggaggga aggcgcgcgc gcgcgcgcgc tcctggaggg gcaccgcagg 661gacccccgac tgtcgcctcc ctgtgccgga ctccagccgg ggcgacgaga gatgcatctt 721cgctccttcc tggtggcggc ggcggctgag aggagacttg gctctcggag gatcggggct 781gccctcaccc cggacgcact gcctccccgc cggcgtgaag cgcccgaaaa ctccggtcgg 841gctctctcct gggctcagca gctgcgtcct ccttcagctg cccctccccg gcgcgggggg 901cggcgtggat ttcagagtcg gggtttctgc tgcctccagc cctgtttgca tgtgccgggc 961cgcggcgagg agcctccgcc ccccacccgg ttgtttttcg gagcctccct ctgctcagcg 1021ttggtggtgg cggtggcagc atggcgagcc ctccggagag cgatggcttc tcggacgtgc 1081gcaaggtggg ctacctgcgc aaacccaaga gcatgcacaa acgcttcttc gtactgcgcg 1141cggccagcga ggctgggggc ccggcgcgcc tcgagtacta cgagaacgag aagaagtggc 1201ggcacaagtc gagcgccccc aaacgctcga tcccccttga gagctgcttc aacatcaaca 1261agcgggctga ctccaagaac aagcacctgg tggctctcta cacccgggac gagcactttg 1321ccatcgcggc ggacagcgag gccgagcaag acagctggta ccaggctctc ctacagctgc 1381acaaccgtgc taagggccac cacgacggag ctgcggccct cggggcggga ggtggtgggg 1441gcagctgcag cggcagctcc ggccttggtg aggctgggga ggacttgagc tacggtgacg 1501tgcccccagg acccgcattc aaagaggtct ggcaagtgat cctgaagccc aagggcctgg 1561gtcagacaaa gaacctgatt ggtatctacc gcctttgcct gaccagcaag accatcagct 1621tcgtgaagct gaactcggag gcagcggccg tggtgctgca gctgatgaac atcaggcgct 1681gtggccactc ggaaaacttc ttcttcatcg aggtgggccg ttctgccgtg acggggcccg 1741gggagttctg gatgcaggtg gatgactctg tggtggccca gaacatgcac gagaccatcc 1801tggaggccat gcgggccatg agtgatgagt tccgccctcg cagcaagagc cagtcctcgt 1861ccaactgctc taaccccatc agcgtccccc tgcgccggca ccatctcaac aatcccccgc 1921ccagccaggt ggggctgacc cgccgatcac gcactgagag catcaccgcc acctccccgg 1981ccagcatggt gggcgggaag ccaggctcct tccgtgtccg cgcctccagt gacggcgaag 2041gcaccatgtc ccgcccagcc tcggtggacg gcagccctgt gagtcccagc accaacagaa 2101cccacgccca ccggcatcgg ggcagcgccc ggctgcaccc cccgctcaac cacagccgct 2161ccatccccat gccggcttcc cgctgctcgc cttcggccac cagcccggtc agtctgtcgt 2221ccagtagcac cagtggccat ggctccacct cggattgtct cttcccacgg cgatctagtg 2281cttcggtgtc tggttccccc agcgatggcg gtttcatctc ctcggatgag tatggctcca 2341gtccctgcga tttccggagt tccttccgca gtgtcactcc ggattccctg ggccacaccc 2401caccagcccg cggtgaggag gagctaagca actatatctg catgggtggc aaggggccct 2461ccaccctgac cgcccccaac ggtcactaca ttttgtctcg gggtggcaat ggccaccgct 2521gcaccccagg aacaggcttg ggcacgagtc cagccttggc tggggatgaa gcagccagtg 2581ctgcagatct ggataatcgg ttccgaaaga gaactcactc ggcaggcaca tcccctacca 2641ttacccacca gaagaccccg tcccagtcct cagtggcttc cattgaggag tacacagaga 2701tgatgcctgc ctacccacca ggaggtggca gtggaggccg actgccggga cacaggcact 2761ccgccttcgt gcccacccgc tcctacccag aggagggtct ggaaatgcac cccttggagc 2821gtcggggggg gcaccaccgc ccagacagct ccaccctcca cacggatgat ggctacatgc 2881ccatgtcccc aggggtggcc ccagtgccca gtggccgaaa gggcagtgga gactatatgc 2941ccatgagccc caagagcgta tctgccccac agcagatcat caatcccatc agacgccatc 3001cccagagagt ggaccccaat ggctacatga tgatgtcccc cagcggtggc tgctctcctg 3061acattggagg tggccccagc agcagcagca gcagcagcaa cgccgtccct tccgggacca 3121gctatggaaa gctgtggaca aacggggtag ggggccacca ctctcatgtc ttgcctcacc 3181ccaaaccccc agtggagagc agcggtggta agctcttacc ttgcacaggt gactacatga 3241acatgtcacc agtgggggac tccaacacca gcagcccctc cgactgctac tacggccctg 3301aggaccccca gcacaagcca gtcctctcct actactcatt gccaagatcc tttaagcaca 3361cccagcgccc cggggagccg gaggagggtg cccggcatca gcacctccgc ctttccacta 3421gctctggtcg ccttctctat gctgcaacag cagatgattc ttcctcttcc accagcagcg 3481acagcctggg tgggggatac tgcggggcta ggctggagcc cagccttcca catccccacc 3541atcaggttct gcagccccat ctgcctcgaa aggtggacac agctgctcag accaatagcc 3601gcctggcccg gcccacgagg ctgtccctgg gggatcccaa ggccagcacc ttacctcggg 3661cccgagagca gcagcagcag cagcagccct tgctgcaccc tccagagccc aagagcccgg 3721gggaatatgt caatattgaa tttgggagtg atcagtctgg ctacttgtct ggcccggtgg 3781ctttccacag ctcaccttct gtcaggtgtc catcccagct ccagccagct cccagagagg 3841aagagactgg cactgaggag tacatgaaga tggacctggg gccgggccgg agggcagcct 3901ggcaggagag cactggggtc gagatgggca gactgggccc tgcacctccc ggggctgcta 3961gcatttgcag gcctacccgg gcagtgccca gcagccgggg tgactacatg accatgcaga 4021tgagttgtcc ccgtcagagc tacgtggaca cctcgccagc tgcccctgta agctatgctg 4081acatgcgaac aggcattgct gcagaggagg tgagcctgcc cagggccacc atggctgctg 4141cctcctcatc ctcagcagcc tctgcttccc cgactgggcc tcaaggggca gcagagctgg 4201ctgcccactc gtccctgctg gggggcccac aaggacctgg gggcatgagc gccttcaccc 4261gggtgaacct cagtcctaac cgcaaccaga gtgccaaagt gatccgtgca gacccacaag 4321ggtgccggcg gaggcatagc tccgagactt tctcctcaac acccagtgcc acccgggtgg 4381gcaacacagt gccctttgga gcgggggcag cagtaggggg cggtggcggt agcagcagca 4441gcagcgagga tgtgaaacgc cacagctctg cttcctttga gaatgtgtgg ctgaggcctg 4501gggagcttgg gggagccccc aaggagccag ccaaactgtg tggggctgct gggggtttgg 4561agaatggtct taactacata gacctggatt tggtcaagga cttcaaacag tgccctcagg 4621agtgcacccc tgaaccgcag cctcccccac ccccaccccc tcatcaaccc ctgggcagcg 4681gtgagagcag ctccacccgc cgctcaagtg aggatttaag cgcctatgcc agcatcagtt 4741tccagaagca gccagaggac cgtcagtagc tcaactggac atcacagcag aatgaagacc 4801taaatgacct cagcaaatcc tcttctaact catgggtacc cagactctaa atatttcatg 4861attcacaact aggacctcat atcttcctca tcagtagatg gtacgatgca tccatttcag 4921tttgtttact ttatccaatc ctcaggattt cattgactga actgcacgtt ctatattgtg 4981ccaagcgaaa aaaaaaaatg cactgtgaca ccagaataat gagtctgcat aaacttcatc 5041ttcaacctta aggacttagc tggccacagt gagctgatgt gcccaccacc gtgtcatgag 5101agaatgggtt tactctcaat gcattttcaa gatacatttc atctgctgct gaaactgtgt 5161acgacaaagc atcattgtaa attatttcat acaaaactgt tcacgttggg tggagagagt 5221attaaatatt taacataggt tttgatttat atgtgtaatt ttttaaatga aaatgtaact 5281tttcttacag cacatctttt ttttggatgt gggatggagg tatacaatgt tctgttgtaa 5341agagtggagc aaatgcttaa aacaaggctt aaaagagtag aatagggtat gatccttgtt 5401ttaagattgt aattcagaaa acataatata agaatcatag tgccatagat ggttctcaat 5461tgtatagtta tatttgctga tactatctct tgtcatataa acctgatgtt gagctgagtt 5521ccttataaga attaatctta attttgtatt ttttcctgta agacaatagg ccatgttaat 5581taaactgaag aaggatatat ttggctgggt gttttcaaat gtcagcttaa aattggtaat 5641tgaatggaag caaaattata agaagaggaa attaaagtct tccattgcat gtattgtaaa 5701cagaaggaga tgggtgattc cttcaattca aaagctctct ttggaatgaa caatgtgggc 5761gtttgtaaat tctggaaatg tctttctatt cataataaac tagatactgt tgatctttta 5821aaaaaaaaaa aaaaaaaaaa aaaaaaaa(SEQ ID NO:6; GENBANK Accession No. NM 005544)

The double mutation of tyrosine 897 and 1180 was constructed byreplacement of 3′-sequences coding 897F by the same region of 1180Fusing restriction enzymes NheI and EcoRI, and this construct was called897F1180F or ΔGrb2 ΔSyp. The expression plasmids were under control of aCMV promoter (hIRS-1-wt, ΔGrb2, ΔSyp, ΔGrb2, ΔSyp and pBK-CMV (mock) andlinearized at the 3′-end of poly A signal sequences by MluI restrictionenzymes followed by purification. A similar approach was used to changethe tyrosine residue to phenylalanine at positions 613 and 942 to createthe double PI3K mutant construct (ΔPI3K). The hIRS-1 mutants have a FLAGepitope (DYKDDDDK (SEQ ID NO:6)+stop codon) added to the C-terminus byPCR. This strategy allows to distinguish the mutant protein from “wildtype” hIRS-1 in stable transfected cell lines. The mutants are used todefine the link between the IRS signal transduction pathway andactivation of HAAH as a downstream effector gene and identify compoundsto inhibit transduction along the pathway to inhibit growth of tumorscharacterized by HAAH overexpression. Antibodies or other compoundswhich bind to phosphorylation sites or inhibit phosphorylation at thosesites are used to inhibit signal transduction and thus proliferation ofHAA-overexpressing tumors.

Other embodiments are within the following claims.

1. A method for diagnosing a malignant neoplasm in a mammal, comprisingcontacting a bodily fluid from said mammal with an antibody or fragmentthereof which binds to an human aspartyl (asparaginyl) beta-hydroxylase(HAAH) polypeptide under conditions sufficient to form anantigen-antibody complex and detecting the antigen-antibody complex,wherein an increase in antigen-antibody complex indicates an increase inHAAH level compared to a normal noncancerous control, said increasebeing indicative of a malignant neoplasm and wherein said antibody is amonoclonal antibody produced by hybridoma ATCC designation
 3386. 2. Themethod of claim 1, wherein said neoplasm is derived from endodermaltissue.
 3. The method of claim 1, wherein said neoplasm is selected fromthe group consisting of colon cancer, breast cancer, pancreatic cancer,liver cancer, and cancer of the bile ducts.
 4. The method of claim 1,wherein said neoplasm is a cancer of the central nervous system (CNS).5. The method of claim 1, wherein said bodily fluid is selected from thegroup consisting of a CNS-derived bodily fluid, blood, serum, urine,saliva, sputum, lung effusion, and ascites fluid.
 6. The method of claim5, wherein said bodily fluid is serum.
 7. A method for prognosis of amalignant neoplasm of a mammal, comprising (a) contacting a bodily fluidfrom said mammal with an antibody which binds to an HAAH polypeptideunder conditions sufficient to form an antigen-antibody complex anddetecting the antigen-antibody complex; (b) quantitating the amount ofcomplex to determine the level of HAAH in said fluid; and (c) comparingthe level of HAAH in said fluid with a normal noncancerous control levelof HAAH, wherein increasing levels of HAAH over time indicates anadverse prognosis, and wherein said antibody is a monoclonal antibodyproduced by hybridoma ATCC designation
 3386. 8. The method of claim 1 or7, wherein said antibody is a single chain Fv molecule.
 9. The method ofclaim 8, wherein said molecule is obtained from an antibody produced byhybridoma ATCC designation PTA
 3386. 10. The method of claim 1 or 7,wherein the antigen-antibody complex is detected by a label selectedfrom consisting of an enzymatic label, a fluorescent label, achemiluminescent label, a radioactive label, and a dye label.
 11. Themethod of claim 1, wherein said neoplasm is a hepatocellular carcinoma.12. The method of claim 1, wherein said neoplasm is acholangiocarcinoma.
 13. The method of claim 1, wherein said neoplasm isa glioblastoma.
 14. The method of claim 1, wherein said neoplasm is aneuroblastoma.
 15. The method of claim 1, wherein said neoplasm is apancreatic cancer.
 16. The method of claim 1, wherein said antibodycomprises a first HAAH-specific antibody and a second HAAH-specificantibody.
 17. The method of claim 1, wherein said tumor is aneuroectodermal tumor.
 18. A method for diagnosing a malignant neoplasmin a mammal, comprising contacting a bodily tissue from said mammal withan antibody or fragment thereof which binds to an human aspartyl(asparaginyl) beta-hydroxylase (HAAH) polypeptide under conditionssufficient to form an antigen-antibody complex and detecting theantigen-antibody complex, wherein an increase in antigen-antibodycomplex indicates an increase in HAAH level compared to a normalnoncancerous control, said increase being indicative of a malignantneoplasm and wherein said antibody is a monoclonal antibody produced byhybridoma ATCC designation
 3386. 19. The method of claim 18, whereinsaid neoplasm is derived from endodermal tissue.
 20. The method of claim18, wherein said neoplasm is selected from the group consisting of coloncancer, breast cancer, pancreatic cancer, liver cancer, and cancer ofthe bile ducts.
 21. The method of claim 18, wherein said neoplasm is acancer of the central nervous system (CNS).
 22. The method of claim 18,wherein said bodily tissue is a solid tumor biopsy.
 23. The method ofclaim 18, wherein said tissue is a histopathological tissue.
 24. Amethod for prognosis of a malignant neoplasm of a mammal, comprising (a)contacting a bodily tissue from said mammal with an antibody which bindsto an HAAH polypeptide under conditions sufficient to form anantigen-antibody complex and detecting the antigen-antibody complex; (b)quantitating the amount of complex to determine the level of HAAH insaid fluid; and (c) comparing the level of HAAH in said tissue with anormal noncancerous control level of HAAH, wherein increasing levels ofHAAH over time indicates an adverse prognosis, and wherein said antibodyis a monoclonal antibody produced by hybridoma ATCC designation 3386.25. The method of claim 18 or 24, wherein said antibody is a singlechain Fv molecule.
 26. The method of claim 25, wherein said molecule isobtained from an antibody produced by hybridoma ATCC designation PTA3386.
 27. The method of claim 18 or 24, wherein the antigen-antibodycomplex is detected by a label selected from consisting of an enzymaticlabel, a fluorescent label, a chemiluminescent label, a radioactivelabel, and a dye label.
 28. The method of claim 18, wherein saidneoplasm is a hepatocellular carcinoma.
 29. The method of claim 18,wherein said neoplasm is a cholangiocarcinoma.
 30. The method of claim18, wherein said neoplasm is a glioblastoma.
 31. The method of claim 18,wherein said neoplasm is a neuroblastoma.
 32. The method of claim 18,wherein said neoplasm is a pancreatic cancer.
 33. The method of claim18, wherein said antibody comprises a first HAAH-specific antibody and asecond HAAH-specific antibody.
 34. The method of claim 18, wherein saidtumor is a neuroectodermal tumor.